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BIOLOGY 

LIBRARY 

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BXORAIK 


THE  RESULTS  OF  SELECTION  WITHIN 

PURE  LINES  OF  PESTALOZZIA 

GUEPINI  DESM. 


BY 


CARL  DOWNEY  LA  RUE 


A  DISSERTATION  SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE 

REQUIREMENTS  FOR  THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY 

AT  THE  UNIVERSITY  OF  MICHIGAN 

MAY,    1921 


Reprinted  from  GENETICS  7:  142-201,  March,  1922 


GENETICS 

A  Periodical  Record  of  Investigations  Bearing  on 
Heredity  and  Variation 


EDITORIAL  BOARD 

GEORGE  H.  SHULL,  Managing  Editor 
Princeton  University 

WILLIAM  E.  CASTLE  EDWARD  JV1.  EAST 

Harvard  University  Harvard  University 

EDWIN  G.  CONKLIN  ROLLINS  A.  EMERSON 

Princeton  University  Cornell  University 

CHARLES  B.  DAVENPORT  HERBERT  S.  JENNINGS 

Carnegie  Institution  of  Washington  Johns  Hopkins  University 

BRADLEY  M.  DAVIS  THOMAS  H.  MORGAN 

University  of  Michigan  Columbia  University 

RAYMOND  PEARL 

Johns  Hopkins  University 


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THE  RESULTS  OF  SELECTION  WITHIN  PURE  LINES  OF 
PESTALOZZIA  GUEPINI  DESM.1 

CARL  DOWNEY  LA  RUE 

University  of  Michigan,  Ann  Arbor,  Michigan 

Received  June  30,  1921 

TABLE  OF  CONTENTS 

INTRODUCTION 1*2 

Reason  for  the  choice  of  Pestalozzia 145 

Experiments 1*0 

Methods  and  conditions 150 

Selection  of  spores  according  to  their  progeny 

Experiment  1.     Selection  for  length  of  spores 158 

Experiment  2.     Selection  for  length  of  spore  appendages 162 

Selection  of  spores  according  to  visible  characters 167 

Experiment  3 168 

Experiment  4 172 

DISCUSSION 174 

CONCLUSIONS 182 

LITERATURE  CITED • 182 

APPENDIX — TABLES 184 

INTRODUCTION 

Since  JOHANNSEN'S  classic  experiments  on  beans  led  him  to  formulate 
his  theory  of  the  multilinear  composition  of  species  and  the  inefficacy 
of  selection  within  any  of  the  lines,  several  workers  have  studied  the  effect 
of  selection  within  pure  lines  of  various  other  organisms.  No  apology  need 
be  offered,  however,  for  this  extension  of  the  work  to  one  of  the  asexually 
reproducing  Fungi  Imperfecti,  for  the  peculiarities  of  the  material  make 
possible  a  quite  different  experimental  attack.  Inasmuch  as  the  literature 
of  the  selection  problem  has  recently  been  thoroughly  reviewed  by 
JENNINGS  (1916,  1920).  and  others,  only  those  papers  will  be  considered 
here  which  have  special  bearing  on  the  present  investigation. 

One  of  the  most  noteworthy  of  such  contributions  is  that  of  JENNINGS 
(1908)  on  Paramecium.  In  a  carefully  planned  and  executed  series  of 
experiments  in  which  numerous  selections  were  made,  JENNINGS  was  unable 

1  Papers  from  the  Department  of  Botany  of  the  UNIVERSITY  OF  MICHIGAN,  No.  186.  A 
portion  of  this  investigation  was  carried  out  at  Kisaran,  Asahan,  Sumatra;  the  remainder  in 
the  Botanical  Laboratory  of  the  UNIVERSITY  OF  MICHIGAN. 

CiEN-ETics  7:     142  Mr  1922 


oA       BIOLOGY 
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SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  143 

to  detect  any  effect  of  selection.  This  was  among  the  first  of  the  consider- 
able series  of  investigations  which  appeared  to  bear  out  the  JOHANNSEN 
theory. 

The  work  of  EWING  (1914  a,  1914  b,  1916)  is  of  particular  interest  be- 
cause of  the  large  number  of  generations  during  which  he  practiced  selec- 
tion. He  studied  an  aphid,  Aphis  avenae,  and  found  that  selection,  even 
for  eighty-seven  generations,  was  totally  without  effect. 

The  work  of  HANEL  (1908)  has  received  much  attention  and  adverse 
comment.  HANEL  concluded  that  selection  was  without  effect  on  Hydra, 
but  it  is  doubtful  whether  the  experimental  results  justified  the  conclusion. 
More  recently  the  status  of  HANEL'S  work  has  been  discussed  and  the  ex- 
periments have  been  repeated  by  L ASHLEY  (1915,  1916).  The  latter 
worker  took  account  of  the  sources  of  error  in  HANEL'S  experiments  and 
carried  out  so  excellent  an  investigation  as  to  leave  no  doubt  that  selec- 
tion within  the  genus  Hydra  is  ineffective. 

A  number  of  other  investigators  have  secured  results  which  substantiate 
the  work  of  JOHANNSEN.  Some  of  them  have  made  careful  investigations 
which  are  of  considerable  value.  In  other  cases  the  results  of  loosely 
planned  and  brief  experiments  have  been  recorded.  For  one  to-  believe, 
because  selection  for  one  generation  within  a  line  has  had  no  effect,  that 
selection  is  therefore  in  all  cases  ineffective,  requires  a  very  sanguine  tem- 
perament. There  was  ground  for  the  protests  of  PEARSON  (1910)  and 
HARRIS  (1911)  that  the  pure-line  theory  had  been  too  readily  accepted 
before  adequate  evidence  had  been  presented.  However,  all  in  all,  a 
considerable  body  of  data  supporting  JOHANNSEN  has  been  secured,  some 
of  it  exceedingly  hard  to  refute.  Until  recently  the  results  of  all  investi- 
gations seemed  only  to  add  more  evidence  in  support  of  the  theory. 

Within  the  last  few  years,  however,  more  and  more  conflicting  evidence 
has  been  amassed,  partly  through  critical  analysis  of  the  older  data,  and 
partly  by  new  experimentation.  JOHANNSEN'S  experiments  have  been 
criticized  because  they  were  not  carried  on  for  a  sufficiently  large  number 
of  generations.  By  statistical  analysis  PEARSON  (1910)  found  that  there 
was  some  evidence  that  JOHANNSEN'S  results  really  showed  inheritance 
of  somatic  variations  within  pure  lines.  ROOT  (1918)  has  discounted 
EWING'S  results  because  the  organism  concerned  is  one  which  in  nature 
is  highly  stable,  its  lack  of  variability  affording  at  the  start  little  ground 
for  hope  that  new  strains  could  be  produced. 

Several  objections  have  been  offered  to  the  conclusions  of  JENNINGS 
from  his  work  on  Paramecium.  One  is  that  he  studied  size  characters 
which  are  highly  variable  under  different  environmental  conditions.  These 

GENETICS  7:    Mr  1922  5  0  7  «J  7  9 


144  CARL  DOWNEY  LA  RUE 

characters  also  vary  with  different  stages  of  maturity  of  the  individuals 
and  it  is  admitted  by  JENNINGS  himself  that  one  cannot  be  certain  that 
only  mature  specimens  are  being  measured. 

Turning  now  from  reinterpretation  of  old  work  to  the  newer  experimen- 
tal data,  we  find  that  several  studies  have  been  interpreted  as  showing 
positive  selection  effects.  Thus  STOCKING  (1915)  found  that  it  was  pos- 
sible to  produce  distinct  groups  by  selection  within  certain  lines  of  abnor- 
mal Paramecia.  MIDDLETON  (1915)  was  able  to  produce  strains  of 
Stylonychia,  distinct  in  regard  to  rate  of  fission,  and  this  almost  at  will. 
JENNINGS  (1916),  with  much  greater  difficulty,  detected  small  changes 
in  DifHugia,  which  he  attributed  to  selection.  ROOT  (1918)  and  HEGNER 
(1919),  working  with  Centropyxis  and  Arcella,  respectively,  secured  data 
in  conformity  with  those  of  JENNINGS  with  DifHugia. 

To  this  later  work,  however,  certain  objections  have  been  made.  STOCK- 
ING'S results  were  obtained  with  abnormal  forms  and  it  seems  to  have  been 
generally  concluded  that  normal  variations  might  not  necessarily  behave  in 
the  same  way.  MAST  (1917)  secured  two  groups  distinct  for  rate  of 
fission  in  Didynium  nasutum,  without  selection,  presumably  by  a  mutation 
in  one  group.  This  has  some  bearing  on  MIDDLETON'S  results,  and  if  it 
be  argued  that  very  frequent  mutations  would  be  needed  to  account  for 
the  rapid  changes  produced  by  MIDDLETON'S  selections,  it  may  be  pointed 
out  that  great  instability  must  obtain  in  an  organism  to  allow  of  such 
changes  even  by  selection. 

MORGAN  (1916)  suggests  that  the  selection  results  with  DifHugia  may 
be  due  to  random  distribution  of  discrete  particles  of  nuclear  material 
(chromidia)  to  the  pairs  of  offspring.  If  such  were  the  case,  and  if  the 
bearers  of  particular  hereditary  characters  were  contained  in  the  chromi- 
dia, one  would  not  expect  the  effects  of  selection  to  be  indefinitely  cumula- 
tive. Moreover,  the  variations  would  necessarily  be  of  a  discontinuous 
type,  and  perhaps  remotely  comparable  to  non-disjunctional  mutations 
or  somatic  mutations.  The  same  suggestion  would  also  apply  to  the 
res'ults  of  selection  in  Centropyxis  and  Arcella.  In  all  three  cases  it  is 
likely  that  the  variations  dealt  with  are  not  of  the  usual  continuous  type. 

LASHLEY  (1915,  1916)  has  produced  evidence  that  PEARSON'S  criticism 
of  JOHANNSEN'S  work  is  not  well  founded,  and  that  the  parent-offspring 
correlation  is  not  reliable  as  a  criterion  in  selection  work. 

It  must  be  obvious  that  the  question  as  to  whether  variations  occurring 
within  a  pure  line  are  purely  somatic  and  non-heritable  or  not,  is  one  that 
is  far  from  a  definite  answer.  Further  investigations  on  forms  suitable 
for  the  study  of  the  problem,  wherever  they  may  occur,  are  greatly  needed. 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  145 

Thus  far  Protozoa  have  been  used  in  most  of  the  critical  and  extensive 
investigations,  and  these  organisms  are  not  only  subject  to  great  environ- 
mental effects,  but  give  rise  to  only  two  offspring  at  a  time, — a  serious  dis- 
qualification for  the  most  accurate  results.  JOHANNSEN'S  original  material, 
the  garden  bean,  was  ideal  from  the  standpoint  of  number  of  offspring, 
but,  reproducing  sexually,  it  offered  a  possibility  of  Mendelian  segregation 
obscuring  the  true  fluctuation  which  it  was  desired  to  study.  The  Fungi 
Imperfecti  seemed  to  afford  an  ideal  material,  combining  the  advantages 
of  numerous  progeny  with  uniparental,  non-sexual  reproduction. 

REASON  FOR  THE  CHOICE  OF  PESTALOZZIA 

P estalozzia  Guepini  Desm.,  one  of  the  Fungi  Imperfecti,  was  chosen  by 
the  writer  as  a  species  unusually  well  suited  for  use  in  the  study  of  the 
selection  problem.  The  species  is  polymorphic,  containing  a  number  of 
different  strains,  so  that  it  is  not  unlikely  that  new  strains  are  being  formed 
at  the  present  time.  It  is  very  easily  grown  in  culture  in  the  laboratory 
and  does  not  require  tedious,  special  methods.  Sporulation  occurs  readily 
in  culture,  and  development  from  the  spore  to  the  fruiting  stage  requires 
only  a  few  days,  so  that  generations  can  be  grown  in  rapid  succession. 
Spores  are  produced  in  enormous  numbers  so  that  more  than  enough  are 
always  available  for  observation.  They  possess  two  characters  that  are 
capable  of  accurate  measurement,  and  a  change  in  color  at  maturity  en- 
ables one  to  tell  with  certainty  which  spores  are  mature  and  which  imma- 
ture. Size  characters  may  therefore  be  studied  without  fear  that  forms 
apparently  small  are  only  immature.  In  addition  to  these  advantages  the 
spores  are  produced,  as  in  all  Fungi  Imperfecti,  entirely  asexually,  so  that 
there  is  no  possibility  that  the  results  are  influenced  by  sexual  phenomena. 

P  estalozzia  Guepini  Desm.  is  widely  distributed  in  the  eastern  tropics, 
where  it  causes  the  gray-blight  disease  of  tea,  and  leaf-spot  diseases  of 
cocoanut,  betel  nut  palm,  African  oil  palm,  Para  rubber,  and  doubtless 
many  other  wild  and  cultivated  plants  of  those  regions.  The  species  con- 
tains a  number  of  forms  which  differ  more  or  less  markedly  from  one  anoth- 
er in  morphological  characters.  Some  of  them  are  likewise  supposed  to 
be  restricted  to  particular  hosts.  Strains  occurring  in  regions  far  removed 
from  areas  known  to  contain  Pestalozzia  Guepini,  and  on  hosts  not  previ- 
ously reported  for  this  fungus,  have  been  described  as  new  species.  This 
is  only  natural,  for  the  species  was  originally  not  closely  defined.  The 
new  species  however,  are  even  less  perfectly  described  than  P.  Guepini,  so 
that  one  gains  nothing  by  using  their  names.  For  the  purposes  of  this 

GENETICS  7:     Mr  1922 


146  CARL  DOWNEY  LA  RUE 

study  the  concept  of  P.  Guepini  employed  is  that  given  in  a  preliminary 
study  of  the  strains  of  the  species  by  LARus  and  BARTLETT  (1922). 

In  this  investigation  the  authors  found  that  from  thirty-five  isolations 
of  Pestalozzia  from  the  Para  rubber  tree,  the  cocoanut  palm,  the  African 
oil  palm,  the  betel  palm,  and  the  tea  plant,  at  least  fourteen  strains, 
distinct  in  regard  to  morphological  characters,  could  be  distinguished. 
More  minute  morphological  studies  would  in  all  probability  have  resulted 
in  the  division  of  some  of  these  strains  into  smaller  distinct  groups.  Cross 
inoculations  might  possibly  have  revealed  variations  in  regard  to  hosts 
infected,  and  physiological  studies  would  almost  certainly  have  shown  that 
some  isolations  differ  physiologically  from  others  in  the  same  morpho- 
logical group.  The  large  number  of  forms  found  within  the  comparatively 
limited  territory  on  the  East  Coast  of  Sumatra,  from  which  the  cultures 
were  secured  suggests  that  an  equally  large  number  of  strains  might  be 
found  in  other  regions,  many  of  them  doubtless  distinguishable  from  those 
already  studied.  The  formation  of  at  least  one  new  strain  by  mutation 
during  the  period  covered  by  the  present  investigation  of  the  effect  of 
selection  leads  to  the  belief  that  other  strains  are  now  being  formed  in 
nature  and  that  P.  Guepini  is  a  species  in  what  may  be  called  a  plastic 
condition.  In  this  respect  it  is  comparable  to  the  organisms  studied  by 
other  recent  workers  who  have  sought  to  find  a  clue  to  the  process  by 
which  evolution  proceeds. 

Only  the  spores'  of  the  fungus  were  studied  minutely  by  LARUE  and 
BARTLETT  (1922)  in  the  investigation  of  variability  in  P.  Guepini  men- 
tioned above.  The  vegetative  characters  and  growth  habits  also  show 
considerable  variation  but  these  are  much  more  limited  in  range  and  more 
difficult  to  measure  than  the  quantitative  characters  of  the  spores,  and 
therefore  were  not  utilized.  For  selection  studies  also,  the  spore  characters 
were  chosen  as  most  suitable  though  other  characters  might*  have  given 
equally  satisfactory  results  had  they  been  more  easily  measured. 

The  spindle-shaped  spores  of  Pestalozzia  Guepini  are  composed  of  five 
cells  of  which  the  three  central  ones  are  smoky  or  black  at  maturity,  while 
the  other  two  are  hyaline.  The  distal  hyaline  conical  cell  normally  bears 
at  its  tip  three  slender,  unjointed,  hyaline  appendages  which  diverge  so 
that  their  tips  are  widely  separated.  Spores  are  occasionally  found  which 
contain  three  or  four  cells,  and  others  which  bear  two  appendages  or 
even  only  one,  but  these  are  apparently  aberrant  forms.  Thus  far  no 
strain  has  been  found  which  is  constant  for  any  of  these  peculiarities, 
and  in  the  cases  where  the  aberrant  spores  have  been  tested,  they  have 
produced  normal  progeny. 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


147 


The  total  range  of  mean  length  of  spores  for  all  the  strains  of  P.  Guepini 
isolated  by  LARUE  and  BARTLETT  (1922)  was  from  19.9ju  to  28.3/z.  Figure 
1  shows  the  polygons  of  variation  in  spore  length  for  a  few  representative 
strains.  Mean  appendage  lengths  of  the  strains  ranged  from  10.9jit  to 
30.0/i.  The  polygons  for  variation  in  appendage  length  are  shown  for 
six  representative  isolations  in  figure  2.  More  complete  data,  which  can- 
not be  reproduced  here,  are  given  in  the  paper  already  cited.  In  view  of 
the  large  number  of  strains  which  exist  within  the  species,  this  fungus  is  a 
very  favorable  organism  for  use  in  making  an  attempt  to  develop  still 
other  strains  by  the  selection  of  variant  spores. 


4-0 


1 


35 
30 
25 
20 
15 
10 


13 


\ 


14    16     18    20   ^^    24   26    28  30   32    34    36    3G 

FIGURE  1. — Polygons  of  variation  in  spore  length  for  six  strains  of  Pestalozzia  Guepini. 
The  ordinates  are  percentages;  the  abscissae,  lengths  in  p. 

It  is  essential  that  any  organism  which  is  to  be  employed  in  the  study 
of  the  selection  problem  be  easy  to  cultivate  and  in  this  respect  P.  Guepini 
is  admirable.  It  will  grow  readily  and  rapidly  on  almost  any  common 
nutrient  agar  and  can  also  be  grown  on  sterilized  leaves,  twigs  and  fruits 
of  its  common  hosts.  In  Sumatra  usually  only  about  four  days  time  was 
needed  to  develop  the  fungus  from  spore  to  the  fruiting  condition.  In 
Michigan  a  slightly  longer  time  was  needed  to  secure  spores  even  when 
the  fungus  was  kept  at  a  temperature  approximately  the  same  as  that  of 
Sumatra.  Strong  light  appears  to  inhibit  the  growth  of  the  young  myceli- 
um, but  exposure  to  light  after  the  mycelium  is  about  three  days  old  seems 
to  hasten  the  production  of  spores. 


GENETICS  7:    Mr  1922 


148 


CARL  DOWNEY  LA  RUE 


When  speculation  begins  the  spores  are  developed  very  rapidly  and  in 
enormous  numbers.  This  is  a  characteristic  exceedingly  valuable  in 
biometrical  studies,  since  the  variates  are  produced  under  as  nearly  identi- 
cal conditions  as  may  be  found  anywhere.  By  measuring  a  sufficient 
number  of  such  spores  one  can  get  the  whole  range  of  variability  induced 
by  the  reaction  of  a  given  set  of  environmental  conditions  with  the  heredi- 
tary characters  of  the  organism.  In  an  organism  which  produces  offspring 
slowly  a  number  of  fluctuations  in  environment  must  necessarily  take  place 


>    10  \l  14  16  16  ?0  II  24  26  28  30  32  54  36      40 


FIGURE  2. — Polygons  of  variation  in  length  of  spore  appendages  for  six  strains  of  Pestalozzia 
Guepini.  The  ordinates  are  percentages;  the  abscissae,  lengths  of  appendages  in  M. 

before  a  number  of  individuals  sufficient  to  give  a  reliable  mean  is  produced. 
This  complicates  the  situation  greatly  and  the  means  of  two  groups  of 
organisms  so  produced  are  less  readily  comparable  than  those  developed 
in  an  organism  such  as  Pestalozzia. 

Mention  has  already  been  made  of  the  dark  color  of  the  three  central 
cells  of  mature  spores  of  P.  Guepini.  This  color  is  a  valuable  index  of  the 
age  of  the  spores  because  it  does  not  fully  develop  until  the  spores  have 
reached  their  full  size  and  the  appendages  have  been  fully  formed.  One 
can  always  recognize  mature  spores  at  a  glance  and  is  thus  able  to  reject 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  149 

all  immature  stages  of  development.  This  completely  obviates  one  great 
difficulty  usually  found  in  the  study  of  size  characters,  that  of  determining 
.which  individuals  are  really  small,  which  only  immature.  This  difficulty 
seems  insurmountable  in  such  forms  as  Paramecium,  but  in  Pestalozzia, 
as  in  Difflugia,  the  nature  of  the  organism  allows  size  characters  to  be 
observed  without  fear  of  complications  from  this  source. 

A  further  word  of  explanation  may  be  desirable  with  regard  to  the 
assumed  absence  of  sexual  reproduction  in  Pestalozzia.  As  has  been  stated, 
it  belongs  to  the  group  of  Fungi  Imperfecti.  Many  of  the  species  in  this 
miscellaneous  assemblage  have  been  found  to  have  other  stages  in  their 
life-history  which  have  enabled  them  to  be  classified  under  other  groups. 
With  few  exceptions  these  other  stages  of  Fungi  Imperfecti  have  shown 
them  to  be  degenerate  members  of  the  Ascomycetes.  Many  Ascomycetes 
have  both  the  ascus  stage,  which  follows  a  sexual  fusion,  and  an  asexual, 
conidial  stage.  It  is  commonly  assumed  that  the  conidial  forms,  which 
make  up  the  Fungi  Imperfecti,  either  have,  or  have  had  at  some  time,  a 
stage  with  a  higher  spore  form.  Whether  Pestalozzia  now  has  an  unknown 
ascus  stage  is  not  so  important,  as  is  the  probability  that  if  it  had  a  sexual 
stage  at  all,  it  would  be  indicated  by  the  appearance  of  a  readily  recogniz- 
able ascus  stage.  No  such  form  has  ever  been  observed  in  the  hundreds 
of  cultures  grown  in  this  series  of  experiments  and  it  is  reasonably  certain 
that  it  never  appeared.  From  the  data  gained  from  the  study  of  analogous 
forms  we  can  safely  assume,  (1)  that  the  conidia  of  Pestalozzia  are  entirely 
asexual,  (2)  that  if  a  sexual  stage  appears  it  will  result  in  the  production 
of  an  ascus  form  and  therefore  will  be  easily  detected,  and  (3)  that  we 
have  no  need  to  fear  that  obscure  nuclear  recombinations  are  complicating 
the  results  secured. 

The  unique  combination  of  characters  possessed  by  Pestalozzia  and 
discussed  above  renders  the  organism  an  unusually  suitable  one  for  use  in 
study  of  the  results  of  selection  within  vegetative  lines.  Because  the 
fungus  was  so  well  adapted  for  this  use  and  because  no  similar  material 
had  ever  been  used  for  this  purpose  the  author  was  led  to  undertake  the 
investigation  here  described.  Presented  in  review,  the  characters  which 
make  Pestalozzia  specially  desirable  for  such  use  are:  (1)  The  presence  of 
numerous  distinct  strains  within  the  species;  (2)  the  ease  with  which  it 
is  grown  in  culture;  (3)  the  rapidity  with  which  consecutive  generations 
may  be  produced;  (4)  the  availability  of  at  least  two  easily  measurable 
independent  characters;  (5)  the  rapidity  with  which  spores  are  produced 
and  the  enormous  numbers  of  spores  produced,  which  enable  one  to  secure 
significant  statistical  constants  for  each  generation;  (6)  the  dark  colora- 

GENETICS  7:     Mr  1922 


150  CARL  DOWNEY  LA  RUE 

tion  of  the  three  central  cells  of  the  spore  which  appears  only  at  maturity, 
and  serves  as  a  criterion  for  the  elimination  of  mere  growth  stages;  (7)  the 
total  absence  of  any  sexual  form  of  reproduction. 

EXPERIMENTS 

Methods  and  conditions 

Since  the  aim  of  the  investigation  was  to  study  variations  of  genetical 
significance,  every  reasonable  attempt  was  made  either  to  remove  varia- 
tions in  environment  or  to  provide  controls  for  them.  All  the  cultures 
used  were  grown  on  agar  in  tubes,  and  under  ordinary  laboratory  condi- 
tions. Aside  from  the  fact  that  it  would  be  extremely  inconvenient,  if  not 
impossible,  to  control  a  selection  experiment  in  which  the  organism  was 
grown  as  a  parasite  on  its  usual  host,  such  a  procedure  would  doubtless 
offer  more  variations  as  to  food  supply,  etc.,  than  would  growth  on  culture 
media.  WOLLENWEBER  (1914)  has  shown  that  organisms  which  are 
facultative  parasites  show  as  normal  development  in  culture  as  on  their 
usual  hosts. 

That  fungi,  especially  Saprolegnias,  may  be  exceedingly  sensitive  to 
variation  in  food  supply  has  been  shown  by  KAUFFMAN  (1908)  and  PIET- 
ERS  (1915).  However  it  is  not  believed  that  Pestalozzia  is  so  exquisitely 
sensitive  to  such  variations  as  are  some  other  fungi.  In  any  event  the 
variations  thus  introduced  into  this  work  are  doubtless  much  smaller  than 
those  which  of  necessity  apply  to  any  other  organism  which  has  been  used 
in  the  study  of  selection  up  to  the  present  time. 

The  nutrient  solution  was  all  made  up  before  the  experiment  was  begun. 
Tender  twig  tips  and  young  leaves  of  Hevea  brasiliensis  were  boiled  in 
water  and  a  quantity  of  brown  sugar  made  from  the  sugar  palm  (Arenga 
saccharifera)  was  added.  The  decoction  was  strained,  concentrated  by 
boiling,  and  after  thorough  mixing  to  secure  uniformity,  was  poured  into 
flasks  which  were  plugged  with  cotton  stoppers  and  sterilized. 

The  single  lot  of  medium  thus  prepared  was  stored  and  used  throughout 
the  experiments  until  the  writer's  return  to  the  United  States.    (A  supply 
of  the  medium  was  brought  to  America  but  was  lost  in  transit,  so  that  it 
was  necessary  to  supply  a  substitute,  as  will  be  noted  later.)    For  growing 
the  cultures  nutrient  agar  was  made  up  according  to  the  following  formula: 
Hevea  decoction,     250  cc 
Water,  3750  cc 

Agar-agar,  120  gm 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  151 

The  medium  was  very  thoroughly  mixed  before  being  put  into  tubes,  and 
the  same  amount  was  placed  in  each  tube.  In  making  cultures  care  was 
taken  that  all  the  tubes  of  a  given  generation  were  poured  from  agar  of 
the  same  lot. 

The  cultures  were  not  kept  in  a  constant-temperature  chamber  but  the 
range  of  variation  in  temperature  and  humidity  is  not  great  in  the  east 
coast  region  of  Sumatra.  For  the  greater  part  of  the  year  the  daily  change 
in  temperature  is  from  about  23°  C  to  about  32°  C.  The  total  range  of 
fluctuation  for  one  five-month  period  of  the  selection  experiment  was  from 
19.4°  to  33.9°  C.  During  the  same  period  the  moisture  content  of  the  air 
varied  from  52  percent  to  100  percent  with  a  usual  daily  range  of  approxi- 
mately from  60  percent  to  100  percent.  In  general  the  climate  of  Asahan 
varies  so  little  from  day  to  day,  or  from  month  to  month,  that  it  would 
be  hard  to  find  any  place  more  uniform.  This  uniformity  allows  cultures 
to  be  carried  on  indefinitely  without  danger  of  extremes  of  heat  or  cold 
such  as  have  ended  various  experiments  of  other  investigators. 

The  selection  experiments  were  made  according  to  two  plans.  In  the 
major  part  of  the  work  the  line  of  descent  was  determined  wholly  by 
divergence  in  progeny  means  without  regard  to  the  visible  characters  of 
the  spores  chosen  as  generation  parents.  In  other  words,  the  direction  of 
selection  in  the  chief  experiments  was  guided  by  the  performance  of  particu- 
lar spores,  rather  than  by  their  appearance,  but  the  method  was  checked 
by  parallel  experiments  in  which  the  usual  basis  of  selection,  namely, 
selection  of  visibly  divergent  individual  spores,  was  employed. 

The  characters  concerned  were  length  of  spore  and  length  of  spore 
appendage.  That  these  characters  are  heritable  to  a  very  considerable 
degree  is  shown  by  the  fact  that  strains,  distinct  for  either  spore  length  or 
for  length  of  spore  appendages,  have  been  isolated,  and  that  these  strains 
remain  distinct  from  generation  to  generation  in  spite  of  fluctuations  in 
environmental  conditions.  The  heritable  differences  between  the  different 
strains  are  very  considerable  as  may  be  seen  from  figures  1  and  2,  and  these 
differences  make  them  especially  promising  for  use  in  a  study  of  the  results 
of  selection. 

The  number  of  cells  in  the  spore  and  the  number  of  appendages  per 
spore  might  appear  to  be  characters  suited  for  study  but  this  was  soon 
found  not  to  be  the  case.  The  number  of  cells  in  the  spore  shows  little 
variation;  the  smallest  number  found  in  observing  hundreds  of  thousands 
of  spores  was  three,  and  the  largest  number  seen  was  nine.  In  two  cases 
where  spores  having  one  more  than  the  normal  number  of  cells  were 

GENETICS  7:    Mr  1922 


152  CARL  DOWNEY  LA  RUE 

selected  it  was  found  that  the  spores  produced  by  the  mycelia  from  these 
spores  were  entirely  normal  in  regard  to  cell  number. 

The  range  of  variation  in  number  of  spore  appendages  is  even  less  than 
that  in  cells.  The  greatest  number  recorded  from  all  observations  made 
was  four  and  the  smallest  number,  one.  Three  different  spores  were  iso- 
lated which  bore  only  one  appendage  each.  However,  when  these  spores 
germinated  and  the  mycelia  from  them  produced  spores,  the  latter  were 
found  to  have  the  normal  three  appendages.  Another  spore  bearing  two 
appendages  was  isolated  with  the  same  result,  and  still  another  bearing 
four  appendages  gave  progeny  which  showed  no  sign  of  this  abnormality. 
Apparently  variation  in  cell  number  and  spore  appendage  number  are  not 
greatly  different  in  behavior  from  sundry  other  abnormalities  which  appear 
from  time  to  time  in  Pestalozzia,  none  of  which  appears  to  be  hereditary. 

Selection  of  spores  according  to  their  progeny 

The  method  of  procedure  in  selecting  spores  according  to  their  progeny 
was  as  follows :  A  single  spore  culture  was  made  from  a  given  strain.  When 
this  culture  produced  mature  spores  agar  plates  were  poured  with  various 
dilution^  of  these  spores.  A  mass  of  spores  was  taken  from  the  culture 
with  a  platinum  wire  and  mixed  with  about  10  ~cc  of  sterile  water  in  a 
sterile  tube.  From  this  water  suspension  of  spores  two  loops  were  put  into 
a  tube  of  melted  agar  and  thoroughly  mixed.  Two  loops  of  this  agar  were 
then  mixed  in  the  next  tube;  and  again  two  loops  were  used  to  inoculate 
the  third  tube.  The  agar  from  each  tube  was  then  poured  into  a  sterile 
petri  dish,  and  the  dish  was  covered  with  a  bell  jar.  When  the  spores 
germinated  the  agar  plates  were  examined  and  spores  were  cut  out  from 
the  most  suitable  ones  and  put  into  separate  tubes  of  Hevea  agar.  Micro- 
scopic examination  was  made  in  each  case  to  make  sure  that  only  one 
spore  was  used  in  inoculating  each  tube.  On  this  account  it  was  found 
more  convenient  to  use  a  1 -percent  beef-extract  agar  instead  of  Hevea 
agar  for  pouring  the  plates.  It  was  not  always  possible  throughout  the 
experiments  to  regulate  the  number  of  spores  in  the  poured  plates.  The 
third  dilution  plate  was  the  one  from  which  the  spores  were  usually  taken, 
but  whenever  this  contained  too  few  spores  to  make  a  full  set  of  cultures 
for  a  given  generation,  the  second  dilution  plate  was  used  instead. 

All  the  tubes  contained  agar  made  in  one  lot,  tubed  and  sterilized  at  the 
same  time,  and  stored  under  the  same  conditions.  The  transfers  of  the 
twenty  single  spores  were  made  one  immediately  after  the  other,  so  that 
the  time  of  transfer  of  each  was  so  nearly  the  same  as  to  admit  of  no 
reasonable  assumption  that  this  variation  could  be  of  effect.  The  twenty 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


153 


tubes  were  put  into  one  rack  so  that  all  might  be  subject  to  the  same 
general  environmental  conditions. 

When  the  mycelia  had  all  grown  and  the  spores  were  mature,  100  spores 
of  each  culture  were  measured  and  the  mean  spore  length,  or  mean  append- 
age length,  as  the  case  might  be,  was  computed.  By  trial  it  was  found 
that  100  measurements  were  sufficient  to  secure  a  smooth  distribution 

TABLE  1 

Typical  frequency  distribution  of  measurements  of  spores  and  spore  appendages  from  different 
cultures  of  Pestalozzia  Guepini.     Measurements  in  (JL. 


SPORE  LENGTHS 

SPORE  APPENDAGE  LENGTHS 

Experi- 

Experiment 1 

Experiment  1 

Experiment  1 

Experiment  1 

Experiment  2 

ment  2 

Length 

Plus  group 

Plus  group 

Minus  group 

Length 

Minus  group 

Plus  group 

Plus 

Generation  3 

Generation  3 

Generation  4 

Generation  6 

Generation  1 

group 

Culture  3 

Culture  8 

Culture  3 

Culture  1 

Culture  1 

Gen.     1 

Cul.    15 

19.8 

1 

2 

3 

10.8 

2 

20.7 

1 

2 

3 

12.6 

2 

2 

21.6 

4 

5 

8 

14.4 

3 

3 

1 

22.5 

8 

9 

11 

16.2 

8 

.    5 

7 

23.4 

11 

17 

15 

18.0 

15 

11 

12 

24.3 

12 

19 

17 

19.8 

17 

12 

15 

25.2 

19 

14 

15 

21.6 

16 

15 

18 

26.1 

14 

12 

12 

23.4 

13 

17 

14 

27.0 

8 

9 

6 

25.2 

10 

14 

10 

27.9 

8 

4 

3 

27.0 

7 

14 

8 

28.8 

8 

4 

2 

28.8 

5 

5 

6 

29.7 

2 

1 

2 

30.6 

2 

3 

3 

30.6 

2 

2 

2 

32.4 

1 

3 

31.5 

1 

1 

34.2 

1 

Means 

25.54±.15 

24.79±.15 

24.53+.  16 

21.22±.21 

22.81±.27 

22.42 
±.31 

Standard 

devia- 

2.18 

.  2.18 

2.38 

4.61 

3.96 

4.57 

tions 

curve  and  a  mean  with  a  satisfactorily  small  probable  error,  so  with  some 
exceptions,  where  a  few  more  spores  were  taken,  one  hundred  from  each 
culture  were  measured.  Typical  distributions  are  shown  in  table  1. 

After  the  twenty  cultures  were  measured  the  one  with  the  greatest  mean 
spore  length  was  chosen  as  the  starting  point  for  plus  selection.  Similarly 
the  culture  with  the  smallest  mean  spore  length  was  taken  as  the  beginning 

GENETICS  7:     Mr  1922 


154 


CARL  DOWNEY  LA  RUE 


of  the  minus  selection.  Another  culture,  which  had  a  mean  spore  length 
as  nearly  midway  between  the  other  two  as  could  be  found,  was  chosen  as 
the  origin  of  an  intermediate  line  to  be  carried  on  from  generation  to 
generation  without  selection. 

From  each  of  the  chosen  cultures  plates  were  now  poured.  When  the 
spores  had  germinated,  ten  single  spores  were  cut  out  from  the  plates 
made  from  the  plus-selection  culture  and  the  same  number  from  the  minus- 
selection  culture.  From  the  intermediate  culture  only  one  single-spore 
culture  was  made.  See  figure  3. 


FIGURE  3.— Plan  of  method  of  selection  used  in  experiments  1  and  2.  A  represents  the 
parent  culture;  B,  the  agar  plate  poured  with  a  dilution  of  spores  from  A;  C,  the  daughter  cul- 
tures grown  from  spores  from  B,  of  which  L  has  the  highest  mean  value  for  the  selected  character 
and  is  chosen  as  parent  of  a  plus-selected  line;  S  has  the  lowest  mean  value  for  the  selected  char- 
acter and  is  selected  as  parent  of  a  minus-selected  line;  and  I,  with  a  mean  as  nearly  intermediate 
between  those  of  L  and  S  as  possible,  is  used  as  parent  of  an  unselected  intermediate  line;  D, 
the  plates  poured  from  cultures  L,  S,  and  I;  and  E,  the  daughter  cultures  grown  from  spores 
isolated  from  the  plates  in  D.  L  and  S  are  again  selected  as  parents  of  the  plus  and  minus 
lines,  respectively,  and  I  is  carried  on  without  selection. 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


155 


All  the  plates  from  the  three  different  cultures  were  poured  on  the  same 
day,  and  from  agar  of  the  same  lot.  All  the  spore  transfers  were  made 
on  the  same  day  and  all  were  kept  in  the  same  rack  under  the  same 
environmental  conditions.  Whatever  the  influences  of  environment  were 
they  would  act  on  all  the  cultures  in  the  same  way,  and  such  variations 
as  might  occur  between  cultures  would  therefore  be  ascribed  to  heredity. 

Some  variation  in  time  of  sporulation  was  always  found  but  no  definite 
correlation  was  noted  between  the  characters  studied  and  the  relative 
rapidity  of  development.  It  was  found  that  the  difference  in  size  between 

TABLE  2 

Comparison  of  spore  appendage  lengths  of  early  and  late  spores  from  the  same  culture.     (Strain 
29,  experiment  2,  generation  17,  plus-selected  group,  culture  3.}     Lengths  in  ju. 


LENGTHS 

EARLY  SPORES 

LATE  SPORES 

9.0 

1 

2 

10.8 

1 

9 

12.6 

4 

13 

14.4 

16 

14 

16.2 

16 

18 

18.0 

28 

23 

19.8 

20 

12 

21.6 

8 

6 

23.4 

2 

3 

25.2 

2 

27.0 

2 

Total 

100 

100 

Means 

18.16±.216 

18.  03  +.234 

Standard  deviations 

3.15+0.15 

3.36±0.16 

Difference  of  means 

0.13+0.32 

the  spores  first  developed  in  a  culture  and  those  developed  several  days 
later  was  not  significant  when  compared  with  the  differences  which  were 
usually  found  between  different  cultures. 

This  fact  is  shown  in  table  2  where  distributions  of  measurements  of 
early  and  late  spores  from  the  same  culture  are  given.  The  early  spores 
were  taken  when  only  a  few  had  been  formed,  the  late  spores  were  the  last 
of  which  the  time  of  formation  was  definitely  known.  It  should  be  noted 
that  all  the  spores  measured  here,  as  in  all  other  parts  of  this  investiga- 
tion, were  mature  normal  spores,  being  fully  septate  and  having  three 


GENETICS  7:     Mr  1922 


156  CARL  DOWNEY  LA  RUE 

dark  central  cells.  The  difference  between  the  mean  appendage  length  of 
early  and  late  spores  is  0.13±0.32ju.  From  SHEPPARD'S  tables  (PEARSON 
1914)  it  may  be  found  that  the  chances  are  fifteen  to  one  that  this  difference 
is  due  to  random  sampling.  Accordingly  it  may  be  assumed  that  the  two 
sets  of  measurements  are  parts  of  the  same  frequency  distribution.  Thus  it 
appears  that  one  may  safely  measure  either  early  or  late  spores  of  a  culture 
without  fear  of  introducing  serious  errors.  In  practice  the  cultures  were 
not  measured  until  a  vast  number  of  spores  had  been  produced  and  the 
sample  was  then  taken  by  drawing  a  sterile  platinum  wire  over  the  fruiting 
surface  of  the  culture  from  the  bottom  of  the  agar  slant  to  its  top.  In  this 
way  a  random  sample  was  doubtless  secured. 

The  sample  was  now  mounted  on  a  clean  slide  on  which  a  drop  of  a 
weak  solution  of  magenta-red  in  40  percent  alcohol  had  been  placed.  The 
spores  were  mixed  in  the  solution  by  stirring  with  the  platinum  wire, 
covered,  and  the  excess  of  solution  removed  with  blotting  paper  after 
which  the  spores  and  appendages  were  clearly  defined  in  the  remaining 
solution.  The  appendages  and  the  hyaline  cells  at  either  end  of  the  spores 
were  stained  deeply  by  the  magenta-red,  so  that  both  spore  length  and 
length  of  appendage  could  be  measured  accurately.  The  solution  did 
not  cause  plasmolysis  or  shrinking  of  either  the  spores  or  the  appendages. 

Duplicate  measurements  were  avoided  by  measuring  all  spores  encoun- 
tered in  a  path  across  the  slide  just  below  the  upper  edge  of  the  cover- 
glass  and  in  another  such  path  just  above  the  lower  edge  of  the  glass. 
The  chances  that  any  spore  was  encountered  twice  were  thus  rendered 
exceedingly  small. 

When  the  means  of  the  ten  cultures  of  the  plus  group  and  those  of  the 
minus  group  had  been  measured  in  the  manner  described  above,  the 
cultures  with  the  longest  and  shortest  means  were  selected  to  continue  the 
plus  and  minus  selections  respectively. 

As  contrasted  with  the  selection  methods  of  other  investigators,  it  will 
be  seen  that  only  the  range  of  variation  happening  to  occur  in  a  random 
sample  of  ten  variates  is  made  available  by  this  method.  It  may  sometimes 
happen  that  ten  variates  chosen  at  random  will  all  fall  near  the  modal 
condition,  but  ten  is  a  large  enough  number  so  that  in  general  a  random 
sample  of  this  size  is  likely  to  spread  over  a  fair  proportion  of  the  range  of 
variation.  To  have  made  more  than  ten  cultures  in  each  line  of  descent  in 
each  generation  would  have  required  an  impossible  amount  of  labor  in 
measuring.  A  larger  number,  as  100,  for  instance,  would  have  insured  the 
detection  of  a  greater  number  of  extreme  variates,  but  would  also  have 
vastly  increased  the  likelihood  of  selecting  sporadic  mutations  rather  than 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  157 

extreme  plus  and  minus  variates  in  the  range  of  continuous  variation. 
From  one  point  of  view  the  method  might  be  looked  upon  as  an  inefficient 
one,  in  that  it  surely  failed  to  take  advantage  of  the  total  range  of  variation 
in  each  generation. 

However,  when  it  is  considered  that  not  only  is  the  culture  from  which 
the  spore  came  known  to  possess  the  quality  for  which  selection  is  being 
made,  but  the  culture  derived  from  each  spore  in  the  line  of  descent  is 
known  to  possess  this  quality  before  that  spore  is  selected  as  a  parent,  it 
is  clear  that  each  selection  is  much  more  significant  than  is  usual  in  cases 
where  many  individuals  are  selected  in  each  generation.  For  example, 
in  the  line  selected  for  plus  spore  length,  each  spore  chosen  as  a  parent 
was  known  to  be  descended  from  a  series  of  ancestral  spores,  each  of  which 
had  the  potentiality  for  producing  a  longer-spored  progeny  than  nine 
other  spores  of  its  own  generation,  chosen  at  random.  Similar  plans  of 
selection  have  been  used  in  one  or  two  previous  investigations  but  not 
extensively  nor  with  so  full  a  knowledge  of  the  behavior  of  both  the 
ancestors  and  the  progeny  of  the  parent  individuals  as  in  the  present  work. 

So  far  as  the  writer  knows  no  other  investigator  of  the  selection  problem 
has  been  able  to  establish  the  mean  of  the  offspring  of  each  selected  indi- 
vidual as  was  done  in  this  experiment.  Most  of  the  organisms  which  have 
been  used  in  selection  experiments  are  of  such  a  nature  as  to  prevent  the 
possibility  of  securing  a  sufficiently  large  number  of  offspring,  all  of  one 
age  and  all  developed  under  the  same  environmental  conditions,  to  estab- 
lish such  means.  Obviously  to  secure  as  many  as  a  hundred  direct  off- 
spring from  one  parent  DifHugia,  (JENNINGS  uses  the  term  parent  as 
applying  to  the  individual  of  a  dividing  pair  which  retains  the  old  shell), 
one  would  have  to  wait  for  just  that  many  divisions  of  the  parent,  which 
would  demand  considerable  time.  The  progeny  being  formed  at  different 
times,  might  be  subjected  to  different  environmental  influences,  so  that  the 
range  of  variation  might  be  greatly  increased.  It  is  therefore  not  possible 
to  know,  in  such  forms  as  Difflugia,  what  the  mean  of  the  offspring  would 
be  were  they  all  produced  under  the  same  conditions.  When  too  small  a 
progeny  is  secured,  one  does  not  know  whether  these  approximate  the 
mean  value  for  the  generation  or  lie  at  one  or  the  other  extreme  of  the 
range.  Pestalozzia  is  especially  favorable  in  that  one  can  attain  a  full 
knowledge  of  each  generation,  which  is  not  the  case  for  most  forms. 
Though  such  data  are  obtained  only  by  the  expenditure  of  a  vast  amount 
of  labor  it  is  believed  they  are  well  worth  while  in  a  serious  study  of  so 
complicated  a  problem  as  that  with  which  we  are  here  concerned. 

GENETICS  7:     Mr  1922 


158 


CARL  DOWNEY  LA  RUE 


Experiment  1.  Selection  for  length  of  spores 

In  this  experiment  the  character  studied  was  the  length  of  the  spore 
from  the  tip  of  the  proximal  cell  to  that  of  the  distal  cell.  In  figure  1, 
frequency  polygons  for  this  character  are  presented  for  six  strains,  of  which 
No.  29  was  the  one  used  in  this  experiment.  Strain  No.  29  was  secured 
from  an  isolation  of  fungi  from  the  wood  of  a  sapling  of  the  Para  rubber 
tree,  Hevea  brasiliensis.  Previous  to  its  use  in  this  experiment  it  had 
been  observed  for  eight  consecutive  generations  in  pure  culture  on  Hevea 
agar.  The  data  concerning  these  generations  are  shown  in  table  3. 

The  experiment  lasted  from  January  1919  to  June  1919  and  included 
ten  selections,  all  of  which  were  made  exactly  according  to  the  method 
described  above.  Through  some  unfavorable  circumstance,  one  of  the 
selections  for  the  eleventh  generation  failed  to  grow  and  the  experiment 
was  thus  brought  to  an  end.  The  entire  experiment  was  done  in  Sumatra, 
and  Hevea  agar  was  used  for  growing  all  the  cultures. 

TABLES 

Mean  spore  lengths  of  cultures  of  strain  29,  grown  prior  to  the  initiation  of  experiment  1.    Mea- 
surements in  //. 


GENERATION 

SPORE  LENGTH 

RANGE  OF  MEA- 

SUREMENTS 

1 

23.6 

21-29 

2 

21.7 

16-27 

3 

25.6 

21-30 

4 

22.2 

17-26 

5 

25.1 

21-29 

6 

25.9 

23-30 

7 

25.5 

21-30 

8 

24.8 

21-29 

9 

25.95 

25-40 

Mean 

24.5 

Figure  4  shows  the  greatest  mean  spore  length  in  each  generation  for  the 
plus  selections  and  the  least  mean  spore  length  in  each  generation  for  the 
minus  selection;  the  so-called  intermediate  line  carried  on  without  selection 
is  shown  also.  The  figure  thus  shows  the  range  of  difference  between  the 
plus  and  minus  selections.  In  case  an  effect  of  selection  were  present, 
this  range  should  become  greater.  In  this  experiment,  this  is  obviously 
not  the  case,  since  the  range  does  not  become  greater.  It  is  true  that  it 
increases  at  different  times  for  a  number  of  generations,  but  this  increase 
is  followed  by  a  corresponding  decrease,  so  that  in  the  end  no  permanent 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


159 


change  is  effected.  The  intermediate  line  shows  continual  fluctuation 
from  generation  to  generation.  Similar  fluctuation  has  been  found  by 
all  workers  who  have  made  careful  studies  of  variation  in  pure  lines  of 
organisms.  It  is  presumably  due  to  environmental  influences,  although 
the  writer  was  unable  to  correlate  very  considerable  fluctuations  in  the 


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FIGURE  4. — Graph  of  the  mean  spore  lengths  of  parent  cultures  of  experiment  1.  P  repre- 
sents the  parents  of  the  plus-selected  line,  M  those  of  the  minus-selected  line,  and  I,  those  of  the 
unselected  intermediate  line.  The  ordinates  are  lengths  in  n;  the  abscissae,  the  generations  of 
the  experiment. 

spore  length  of  Pestalozzia  with  any  significant  fluctuations  in  any  of  the 
environmental  factors  which  are  easily  measured.  The  fluctuations  in 
the  plus  and  minus  selections  appear  to  be  due,  not  to  selection,  but  to 
such  influences  as  cause  the  variations  in  the  unselected  intermediate  line. 
In  most  cases  the  fluctuations  of  the  plus  and  the  minus  selections  in  a 


GENETICS  7:     Mr  1922 


160 


CARL  DOWNEY  LA  RUE 


given  generation  parallel  each  other.  When  this  is  the  case  the  deviation 
between  the  two  selections  is  small.  When  the  fluctuations,  for  any  reason, 
do  not  parallel  each  other  the  deviations  between  the  two  selections  may 
be  considerably  increased  or  considerably  decreased. 

From  an  inspection  of  figure  4  lit  is  difficult  to  tell  whether  or  not  the 
parent  cultures  of  the  plus  and  minus  strains  are  really  different.  It  is 
absolutely  essential  in  selection  studies  that  all  selections  be  significant, 
that  is,  the  plus  selection  must  be  greater  than  the  minus  selection,  not 
only  apparently,  but  statistically.  It  is  by  no  means  certain  that  this 
has  always  been  the  case  in  previous  investigations,  and  in  many  cases 


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FIGURE  5. — Graph  of  means  of  spore  lengths  of  generations  in  experiment  1.  The  plus- 
selected  line  is  designated  by  P;  the  minus-selected  line  by  M.  The  ordinates  are  lengths  in  /u; 
the  abscissae,  the  generations  of  the  experiment. 

it  is  difficult  if  not  impossible  to  know  whether  or  not  the  differences  are 
significant,  especially  where  individuals  are  selected  without  knowledge  of 
the  mean  of  the  generation. 

Pestalozzia  is  a  very  favorable  organism  in  this  respect  since  one  can 
employ  the  mean  spore  length  of  the  selected  cultures  and  know  at  the 
same  time  the  means  of  all  the  other  cultures  of  that  generation,  as  well 
as  the  mean  of  those  means.  In  this  experiment  it  was  found  by  computing 
the  errors  of  the  differences  between  the  plus  and  the  minus  selections  that 
these  differences  were  significant  in  all  cases.  The  smallest  difference  found 
in  the  course  of  the  experiment,  .900  +  .206^  in  the  ninth  selection,  is  more 
than  four  times  its  probable  error  and  may  be  considered  significant. 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


161 


The  means  of  the  different  generations  of  the  plus  and  minus  lines  are 
plotted  in  figure  5  and  show  variations  comparable  to  those  shown  in 
figure  2.  If  an  effect  of  selection  had  been  secured  the  means  must  neces- 
sarily have  shown  increasing  deviation  one  from  another  throughout  the 
experiment.  Such  increases  as  they  have  shown  have  been  compensated 
by  corresponding  decreases  so  that  it  becomes  evident  as  the  experiment 
is  carried  on  for  a  considerable  period  that  these  increases  and  decreases 
of  the  deviation  between  the  two  selections  are  only  the  result  of  the  chance 
fluctuations  which  take  place  within  the  two  lines.  It  is  probable  that  such 

TABLE  4 

Mean  spore  lengths  of  generations  of  experiment  1.  Each  mean  was  computed  from  the  means 
of  the  cultures  in  its  group  for  that  generation.  Each  mean,  except  in  the  intermediate  group  is 
accordingly  based  on  approximately  1000  individual  measurements.  Values  are  given  in  fi. 


GENERATIONS 

PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

1  (unselected) 

24.91 

24.91 

24.91 

2 

25.54 

25.38 

24.12 

3 

25.25 

24.61 

26.59 

4 

24.41 

24.28 

24.38 

5 

24.51 

24.82 

25.25 

6 

24.75 

24.70 

23.94 

7 

24.71 

24.64 

23.83 

8 

24.50 

24.21 

24.23 

9 

24.19 

24.43 

23.92 

10 

24.41 

23.67 

24.03 

Means  of  generation 

means  based  on  means 

24.727  +  0.0559 

24.622  +  0.0407 

24.552±0.126 

of  all  cultures  in  each 

line 

Difference  between  plus-selected  line  and  minus-selected  line  =  0.105 +  0.068. 
Difference  between  plus-selected  line  and  intermediate  line  =  0.18 +  0.104. 
Difference  between  minus-selected  line  and  intermediate  line =0.07 +0.097. 

differences  would  occur  between  any  two  separate  lines,  descended  from 
the  same  parent  but  cultivated  separately. 

The  means  of  each  of  the  generations  in  the  plus-selected  line,  the  minus- 
selected  line,  and  the  unselected  intermediate  group,  and  also  the  mean 
of  means  of  all  cultures  in  each  of  these  lines  for  the  whole  experiment  are 
shown  in  table  4,  while  table  12  (in  the  appendix)  gives  the  means  of  all 
cultures  measured  in  this  experiment.  The  differences  between  the  lines 
are  in  themselves  very  small  and  when  compared  with  their  probable 
errors  they  are  seen  to  be  insignificant.  However,  had  the  experiment 

GENETICS  7:     Mr  1922 


162  CARL  DOWNEY  LA  RUE 

been  discontinued  after  three  selections  it  would  have  appeared  that  a 
small,  but  possibly  significant,  result  had  been  produced  by  the  selections. 
The  difference  between  the  two  lines  at  that  time  was  0.267/1,  which  is 
probably  more  than  three  times  its  probable  error  which  has  not  been 
computed.  It  is  at  any  rate,  more  than  three  times  the  error  of  the  differ- 
ence between  the  plus-  and  minus-selected  lines  for  the  whole  experiment. 

When  selection  is  continued  for  a  longer  time  the  apparent  effect  of 
selection  is  soon  lost  and  the  minus-selected  line  at  times  produces  longer 
spores  than  the  plus-selected  one.  The  intermediate  form,  which  is  entire- 
ly unselected,  shows  greater  fluctuations  than  either  of  the  other  groups, 
and  its  mean  for  the  whole  experiment  is  less  than  that  of  the  minus- 
selected  line.  If  we  assume  that  the  upward  and  downward  swings,  so 
prominent  in  this  experiment,  are  merely  chance  fluctuations  due  to 
environmental  influences  we  can  understand  why  the  intermediate  line 
shows  more  fluctuation  than  the  others,  since  it  is  represented  by  only  one 
culture  in  each  generation  and  its  generation  means  are  computed  from 
only  100  measurements  instead  of  1000  as  in  the  case  of  the  other  two  lines. 

Since  the  two  selected  lines  cannot  be  shown  to  differ  from  one  another, 
or  from  the  unselected  line,  we  can  only  conclude  that  selection  has  been 
of  no  avail  in  producing  lines  of  Pestalozzia  Guepini  distinct  for  length  of 
spore. 

Experiment  2.    Selection  for  length  of  spore  appendages 

In  this  experiment  the  same  strain  was  used  as  in  experiment  1 ,  namely, 
No.  29.  Table  5  presents  the  means  and  ranges  of  appendage  length  for 
the  eight  generations  during  which  the  fungus  had  been  studied  prior  to 
the  initiation  of  this  experiment,  and  a  frequency  polygon  of  this  strain 
is  shown  in  figure  2. 

The  plan  of  the  experiment  was  that  explained  earlier  in  this  paper 
and  used  in  experiment  1,  and  was  rigidly  followed  throughout  the  whole 
series  of  selections  which  were  continued  for  more  than  a  year.  All  of  the 
cultures  were  grown  on  Hevea  agar,  made  according  to  the  formula  already 
given,  from  the  original  lot  of  Hevea  decoction,  and  used  with  all  the 
precautions  previously  mentioned.  This  work,  like  that  of  experiment  1, 
was  all  done  in  Sumatra  within  a  few  hundred  yards  of  the  place  where 
line  No.  29  was  originally  found. 

In  this  experiment  twenty-five  selections  were  made  for  length  of  spore 
appendages,  and  twenty-five  successive  generations  were  grown  of  an 
unselected  intermediate  line  also.  The  appendage  lengths  of  all  the  parent 
spores  are  shown  in  figure  6.  The  extreme  cultures  of  the  plus  and  minus 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


163 


lines  parallel  each  other  in  a  remarkable  manner  but  the  differences 
between  them  are  not  great.  In  one  case  (generation  11)  the  two  lines  are 
so  nearly  coincident  that  parent  cultures  certainly  different  for  appendage 
length,  could  not  be  secured  from  the  plus  and  minus  lines.  The  range 
of  variation  in  appendage  lengths  did  not  consistently  increase  from  gen- 
eration to  generation  as  they  should  if  selection  were  effective  but  under- 
went successive  increases  and  decreases  as  the  two  lines  drew  nearer  to- 
gether or  deviated  more  widely.  The  differences  between  the  plus-selected 
parent  and  the  minus-selected  parent  vary  from  generation  to  generation, 
but  are  large  enough  to  be  of  significance  except  in  the  one  case  mentioned. 
Figure  7  shows  the  means  of  the  generations  for  the  whole  experiment 
and  from  this  figure  it  is  at  once  apparent  that  the  two  lines  of  descent  did 

TABLE  5 

Mean  lengths  of  spore  appendages  of  cultures  of  strain  29,  grown  prior  to  beginning  experiment  2. 

Measurements  in  fi. 


GENERATIONS 

LENGTHS  OF 
APPENDAGES 

RANGE  OF  MEA- 
SUREMENTS 

1 

24.5 

20-30 

2 

21.7 

10-27 

3 

23.1 

16-30 

4 

26.7 

20-36 

5 

25.1 

20-39 

6 

22.4 

14-39 

7 

24.0 

16-33 

8 

24.1 

19-31 

9 

21.8 

16-40 

Mean 

23.71 

not  become  more  widely  divergent  as  the  number  of  selections  increased. 
Instead  the  same  upward  and  downward  swings  appear  as  were  seen  in 
experiment  1,  but  in  this  experiment  the  two  lines  parallel  each  other  in 
these  fluctuations  in  a  surprising  way. 

The  means  of  the  generations  of  each  line  and  the  mean  of  means  of 
each  line  for  the  whole  experiment  are  presented  in  table  6,  while  table  13 
(in  the  appendix)  gives  the  data  for  all  the  cultures  grown  during  the  course 
of  the  experiment.  After  examining  figure  7  one  is  not  surprised  to  find 
that  the  differences  between  the  three  different  experimental  lines  are  of  no 
significance.  In  one  case  the  difference  is  less  than  its  probable  error.  Each 
case  where  the  plus  line  has  grown  longer  and  the  minus  line  shorter  has 


GENETICS  7:     Mr  1922 


164 


CARL  DOWNEY  LA  RUE 


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SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


165 


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GENETICS  7:    Mr  1922 


166 


CARL  DOWNEY  LA  RUE 


apparently  been  so  nicely  balanced  by  one  where  the  minus  line  has  grown 
longer  and  the  plus  line  shorter,  that  the  result  has  been  an  almost  absolute 
identity  of  the  two  lines  as  regards  length  of  spore  appendages. 

TABLE  6 

Mean  appendage  lengths  of  generations  of  experiment  2.    Each  mean  was  computed  from  the 
means  of  the  cultures  in  its  group  for  that  generation.    Each  mean,  except  in  the  intermediate  group, 
is  based  on  approximately  1000  measurements.    Measurements  are  given  in  JJL. 


GENERATIONS 

PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

1 

23.94 

23.94 

23.94 

2 

21.22 

20.54 

23.04 

3 

19.06 

18.74 

20.78 

4 

19.66 

20.74 

20.93 

5 

21.78 

19.73 

20.78 

6 

20.77 

21.0)8 

22.63 

7 

21.32 

21.99 

22.41 

8 

20.88 

21.22 

20.98 

9 

18.61 

18.23 

18.83 

10 

17.33 

17.87 

17.05 

11 

19.71 

21.37 

21.39 

12 

19.42 

19.28 

20.14 

13 

18.99 

18.74 

18.67 

14 

20.66 

21.42 

18.59 

15 

20.90 

20.63 

21.24 

16 

20.61 

19.26 

20.47 

17 

18.56 

18.34 

18.62 

18 

17.98 

18.70 

19.44 

19 

20.68 

20.83 

20.47 

20 

19.62 

19.62 

19.42 

21 

20.03 

19.53 

18.65 

22 

20.20 

20.65 

19.04 

23 

20.34 

20.29 

21.83 

24 

20.74 

20.02 

21.06 

25 

19.31 

19.22 

19.62 

Means     of     generation 

means  based  on  means  of 

20.394+0.0844 

20.223  +  0.0774 

20.502±0.227 

all  cultures  in  each  line 

Standard  deviations 

1.908±0.0598 

1.746±0/050 

1.681+0.160 

Difference  between  plus-selected  line  and  minus-selected  line =0.171 +0.1 15. 
Difference  between  plus-selected  line  and  unselected  intermediate  line  =  0.108  ±0.242. 
Difference  between  minus-selected  line  and  unselected  intermediate  line =0.279 +0.239. 

The  minute  scrutiny  of  each  generation  in  the  course  of  this  experiment 
and  of  experiment  1  made  it  possible  to  detect  at  once  the  appearance  of 
any  aberrant  form  by  mutation.  It  is  desirable  that  such  control  be  kept 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  167 

in  all  selection  studies  since  it  is  not  improbable  that  in  some  cases  small 
mutations  have  arisen  in  the  course  of  selection  experiments,  which  have 
not  been  detected  and  have  produced  effects  attributed  to  selection.  In 
this  experiment  in  the  minus  line  of  generation  14,  culture  5,  a  distinct 
mutation  appeared  which  had  a  greater  length  of  spore  appendages 
than  any  other  culture  encountered  in  the  whole  experiment.  The  spores 
themselves  were  also  longer,  much  more  slender,  and  lighter  in  color  than 
those  of  any  other  culture  seen  during  the  whole  range  of  my  experience 
with  Pestalozzia.  This  form  has  since  remained  distinct  through  several 
generations  and  will  be  studied  further  at  a  later  date.  It  is  mentioned 
here  merely  to  show  the  possibility  that  such  mutations  may  account  for 
some  of  the  results  which  are  apparently  due  to  selection.  In  the  present 
instance  the  mutation  occurred  in  a  plus  direction  but  in  the  minus  line 
and  so  could  hardly  have  escaped  attention,  but  had  it  occurred  in  the 
plus  line,  had  it  been  somewhat  less  striking,  and  had  it  occurred  in  a  less 
carefully  controlled  experiment  it  might  have  escaped  detection,  and  have 
greatly  confused  the  results  of  that  experiment. 

That  the  new  form  was  not  a  contamination  from  outside  instead  of  a 
mutation,  was  made  practically  certain  because  no  such  form  had  ever 
been  isolated  from  the  surrounding  country;  because  all  culture  work 
was  carefully  done  and  the  usual  contaminating  organisms  were  rarely 
found  in  the  cultures;  because  Pestalozzia  is  not  a  fungus  which  is  likely 
to  be  found  as  an  air-borne  contamination  in  cultures;  and  finally,  because 
blank  plates  were  poured  in  many  generations  without  ever  securing  any 
growth  of  any  organism. 

Experiment  2,  carried  on  for  more  than  one  year,  through  twenty-five 
generations,  involving  nearly  500  different  cultures,  and  50,000  spore 
measurements,  should  have  been  a  sufficiently  thorough  and  long-con- 
tinued study  to  demonstrate  any  effect  of  selection  which  might  have  ap- 
peared. Since  no  such  effect  has  been  demonstrated  we  can  only  conclude 
that  the  result  of  this  experiment  agrees  entirely  with  that  of  experiment  1 
and  that  it  is  not  possible  to  produce  lines  distinct  for  lengths  of  spore 
appendages  by  even  long-continued  selection. 

Selection  of  spores  according  to  visible  characters 

The  selection  of  spores  according  to  their  appearance  was  now  under- 
taken as  a  check  on  the  results  of  the  previous  selections.  On  account  of 
the  small  size  of  the  spores  of  Pestalozzia  considerable  difficulty  was  ex- 
perienced in  finding  a  method  by  which  the  measured  spores  could  be 

GENETICS  7:    Mr  1922 


168  CARL  DOWNEY  LA  RUE 

isolated.  Finally  a  special  method  was  devised  which  has  been  described 
in  the  Botanical  Gazette  (LARuE  1920). 

In  this  method  a  selecting  device  was  used  which  consisted  of  a  brass 
cone  threaded  at  one  end,  and  turned  into  a  small  tube  at  the  other,  the 
end  of  the  wall  of  the  tube  being  made  thin  so  as  to  form  a  sharp  cutting 
edge.  The  device  was  screwed  into  the  nosepiece  of  a  microscope  in  place 
of  one  of  the  objectives.  The  spores  to  be  examined  were  mixed  in  a 
sufficient  dilution  in  a  tube  of  1 -per  cent  LIEBIG'S  beef -extract  agar  and 
sterile  slides  were  spread  with  a  thin  layer  of  this  agar.  When  the  agar 
cooled  the  spores  were  firmly  fixed  in  a  thin  transparent  agar  matrix  and 
could  easily  be  located  and  measured  under  the  microscope.  When  a 
spore  of  the  desired  size  was  located  it  was  carefully  centered  in  the  field 
of  vision  and  the  nose-piece  of  the  microscope  was  turned  so  as  to  place 
the  selecting  device,  of  which  the  tip  had  just  been  sterilized  by  flaming 
with  a  gas  or  alcohol  flame,  immediately  above  the  spore.  In  a  good 
microscope,  made  so  that  the  objectives  center  properly,  this  is  an  entirely 
mechanical  process  requiring  no  skill  on  the  part  of  the  operator.  The 
microscope  tube  was  now  lowered  until  the  sharp  tube  entered  the  agar 
and  cut  a  ring  completely  around  the  spore.  The  agar  disk  containing  the 
spore  was  now  examined  to  see  that  only  the  chosen  spore  was  contained 
in  it,  and  then  lifted  with  a  flattened  platinum  wire  and  transferred  to  a 
tube  of  nutrient  agar. 

The  agar  used  for  imbedding  the  spores  on  the  slides  was  filtered  very 
carefully  to  render  it  as  transparent  as  possible  so  that  it  might  not  inter- 
fere with  a  clear  view  of  the  spores.  After  selection  the  spores  were  grown 
in  Hevea  agar  of  the  same  composition  as  that  used  in  the  former  experi- 
ments. However,  after  five  selections  had  been  made  the  cultures  were 
transferred  from  Sumatra  to  Michigan,  and  the  supply  of  Hevea  decoction 
taken  with  them  having  been  lost  in  transit,  prune-juice  agar  was  used 
for  the  remaining  cultures  of  the  experiment.  The  prune  agar,  which 
gave  satisfactory  results,  was  made  by  adding  2  percent  of  agar-agar  to  a 
decoction  of  prunes.  Since  only  one  lot  was  made,  and  used  for  all  the 
cultures  the  exact  proportions  are  of  no  special  significance  in  the  experi- 
ment, and  are  therefore  omitted.  Care  was  taken,  however,  to  see  that  the 
medium  was  as  uniform  as  possible  for  all  the  cultures. 

Experiment  3 

The  line  used  for  this  experiment  was  No.  17,  an  isolation  made  from  a 
leaf-spot  of  a  seedling  of  Hevea  brasiliensis,  which  had  been  grown  in 
culture  and  measured  for  nine  generations  prior  to  the  initiation  of  the 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


169 


experiment.    The  variation  of  the  strain  during  these  generations  may  be 
seen  in  table  7. 

The  character  selected  in  this  study  was  length  of  spore.  Spores  of  the 
last  culture  shown  in  table  7  were  mounted  in  agar  on  slides  and  measured. 
The  longest  spore  found  was  selected  as  the  parent  of  a  plus-selected  line, 
*  and  the  shortest  spore  encountered  was  chosen  as  the  parent  of  a  minus- 
selected  line.  When  sporulation  took  place  in  the  resultant  cultures 
the  spores  were  measured  and  the  mean  spore  lengths  of  the  cultures 
determined  precisely  as  in  experiments  1  and  2.  Spores  from  each  culture 
were  now  mounted  and  measured,  the  longest  found  in  the  plus  cul- 

TABLE  7 

Mean  spore  lengths  of  cultures  of  strain  17,  grown  prior  to  the  selections  in  experiment  3.  Mea- 
surements in  (JL. 


GENERATIONS 

SPORE  LENGTHS 

RANGE  OF  MEA- 

SUREMENTS 

1 

22.0 

17-24 

2 

21,1 

17-23 

3 

21.2 

19-24 

4 

23.4 

20-29 

5 

22.3 

19-29 

6 

22.5 

17-26 

7 

23.3 

19-27 

8 

23.5 

20-28 

9 

23.9 

20-28 

Means 

22.58 

ture  being  selected  to  continue  the  plus  line,  and  the  shortest  found  in  the 
minus  culture  being  taken  as  parent  of  the  next  generation  of  the  minus 
line. 

The  selections  thus  begun  were  continued  until  ten  had  been  made 
when  the  experiment  was  discontinued.  No  attempt  was  made  to  make 
the  selections  in  the  two  lines  at  the  same  time.  Thus  the  cultures  in  the 
two  groups  usually  developed  under  somewhat  variable  environmental 
conditions  so  that  a  given  selection  is  not  precisely  comparable  with  the 
selection  of  the  same  number  in  the  other  line.  However,  in  the  long  run 
this  variation  is  probably  not  significant  since  the  fluctuations  in  environ- 
ment should  be  about  the  same  in  summation  for  one  line  as  for  the  other. 


GENETICS  7:     Mr  1922 


170 


CARL  DOWNEY  LA  RUE 


From  the  nature  of  the  method  of  selection,  spores  near  the  extremes 
of  the  range  of  variation  were  chosen,  and  accordingly  the  difference 
between  the  selected  spore  of  the  minus  line,  and  that  for  the  plus  line  in  a 
given  generation,  was  always  large.  The  lengths  of  the  selected  spores 
for  the  experiments  are  shown  in  figure  8.  The  differences  between  the 
parent  spores  of  the  two  groups  does  not  increase  significantly  during  the 
experiment,  nor  does  it  decrease.  At  the  time  the  cultures  were  transferred 
to  America  the  lengths  of  the  parent  spores  decreased  considerably  but  the 
deviations  between  them  were  unchanged.  To  what  extent  the  decrease 
in  spore  length  was  due  to  change  of  culture  medium,  and  to  what  extent 
due  to  change  of  climate,  is  unknown. 


30 

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1ft 

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\ 

M 

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x 

N 
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— 

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X. 

M 

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) 

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t 

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FIGURE  8. — Graphs  of  the  lengths  of  individual  spores  selected  as  parents  of  the  plus  and 
minus  line  of  experiment  3.  P,  the  plus  selections;  M,  the  minus  selections.  The  ordinates  are 
lengths  in  /*;  the  abscissae,  the  successive  generations. 

The  mean  spore  lengths  of  the  generations  of  this  experiment  are  shown 
in  table  8.  The  removal  of  the  cultures  to  America  resulted  in  a  decrease 
in  mean  spore  length  which  is  clearly  shown  in  figure  9.  The  plus  and 
minus  lines  did  not  parallel  each  other  so  closely  as  in  experiments  1  and  2. 
This  is  to  be  expected  because  the  selections  in  the  two  lines  were  not 
usually  made  at  the  same  time.  The  two  lines  were  consequently  not  in 
harmony  in  regard  to  the  conditions  under  which  they  were  developed  and 
their  fluctuations  were  not  parallel.  In  five  of  the  ten  generations  the 
minus  line  has  a  greater  spore  length  than  the  plus-selected  one.  That 
this  result  is  due  to  selection  is  unthinkable.  It  serves  as  a  warning  that 
small  changes,  apparently  in  the  direction  of  a  selection  effect,  should  not 
too  readily  be  considered  as  due  to  selection,  though  they  have  usually 
been  so  interpreted. 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


171 


The  means  of  means  of  all  the  cultures  in  the  two  lines  when  compared 
are  found  not  to  be  significantly  different.  The  upward  and  downward 
swings  have  had  the  same  total  effect  in  one  line  as  in  the  other  though  the 
different  lines  have  scarcely  paralleled  each  other  at  all  during  these  swings. 
Experiment  3,  then,  entirely  substantiates  the  results  gained  in  experi- 
ments 1  and  2,  and  shows  that  the  selection  of  visible  characters  within  a 
pure  line  is  no  more  effective  in  producing  distinct  groups  than  selection 
on  the  basis  of  progeny. 

In  the  course  of  experiment  3  an  aberrant  form  appeared,  which  seems 
to  be  a  mutation.  This  arose  in  the  plus  line  in  the  seventeenth  generation. 


25 
24 
23 
22 
21 
20 

M 

\," 

-£ 

/ 
/ 

f^ 

V 
\ 
\ 

,'*\. 

/ 

\\ 

1 

—  — 

""/ 

*s* 

\ 

^ 

Ss 

\ 

/ 

\ 

/ 

X 

> 

f 

2345          67         69        10 

FIGURE  9. — Graphs  of  the  spore  lengths  of  cultures  grown  in  experiment  3.  P  shows  the 
cultures  of  the  plus  line;  M  those  of  the  minus  line.  The  ordinates  are  lengths  in  n;  the  abscissae, 
the  generations  of  the  experiment. 

In  regard  to  length  of  spores  and  of  spore  appendages  it  is  identical  with 
strain  17  from  which  it  arose.  However,  it  has  vegetative  characters 
quite  distinct  from  those  of  strain  1 7  and  this  makes  it  appear  very  different 
to  the  naked  eye.  Strain  17  produces  a  very  small  amount  of  mycelium 
on  the  surface  of  the  agar  substratum,  which  becomes  almost  completely 
covered  with  a  black,  slimy  mass  of  spores.  The  new  form  produces  a 
thick  felt  of  mycelium  all  over  the  surface  of  the  agar,  and  sporulation  is 
much  more  tardy  than  in  strain  17.  The  spores  are  produced  in  small 
black  masses  which  are  rather  sparsely  scattered  over  the  surface  of  the 


GENETICS  7:    Mr  1922 


172 


CARL  DOWNEY  LA  RUE 


mycelium.  Thus  far  the  form  has  remained  distinct  for  two  generations, 
and  will  be  studied  further  in  the  near  future.  If  it  continues  to  show 
distinctive  characters  it  will  have  to  be  considered  as  a  mutation,  though 
one  which  affected  fewer  characters  than  the  one  encountered  in  experi- 
ment 1.  More  detailed  statements  concerning  both  these  mutations  will 
be  presented  in  another  publication. 

TABLE  8 

Mean  spore  lengths  of  cultures  grown  in  experiment  3.     One  culture  was  grown  in  each  line 
in  each  generation.    Each  mean  is  based  on  100  measurements.    Measurements  in  fj,. 


GENERATIONS 

PLUS  SELECTIONS 

MINUS  SELEC- 

TIONS 

1 

24.37 

23.65 

2 

23.53 

24.30 

3 

24.34 

24.16 

4 

24.01 

25.04 

5 

22.09 

22.30 

6 

20.78 

22.40 

7 

22.56 

22.46 

,       8 

21.43 

22.98 

9 

22.58 

22.51 

10 

21.97 

21.76 

Means  of 

generation 

22.  766  +.251 

23.16+.  212 

means 

Standard 

deviations 

1.18+.17 

1.01  +  .15 

Difference  between  plus-selected  line  and  minus-selected  line =0. 390 +0.328/-1. 

Experiment  4 

It  was  originally  intended  that  this  experiment  should  be  carried  on  for 
an  extended  period  of  time.  In  order  to  lessen  the  labor  required,  some 
changes  were  made  in  the  method  which  had  been  used  in  experiment  3. 
The  spores  were  selected  and  grown  in  the  same  manner  as  in  experiment  3 
but  the  cultures  of  each  generation  were  not  measured.  Instead  it  was 
planned  to  measure  them  only  from  time  to  time  to  determine  whether 
or  not  the  plus  and  minus  lines  were  diverging  one  from  another.  Un- 
fortunately only  a  few  selections  had  been  made  before  the  writer  left 
Sumatra.  The  strain  used,  No.  5,  which  was  isolated  from  a  leaf  spot  of 
cocoanut  palm  and  had  been  successfully  grown  for  nine  generations  in 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


173 


culture  with  the  results  shown  in  figures  1  and  2,  and  in  table  9,  did  not 
thrive  well  in  Michigan,  even  when  kept  in  a  constant-temperature  room, 
so  after  six  selections  the  experiment  was  discontinued.  While  the  experi- 
ment included  fewer  selections  than  is  desirable,  a  large  number,  perhaps 
the  majority,  of  selection  experiments  have  been  carried  no  farther,  and 
no  apology  need  be  made  for  presenting  the  results.  These  are  shown  in 
table  10  and  the  measurements  of  the  selected  parent  spores  are  presented 
in  figure  10. 

The  spores  selected,  on  the  basis  of  spore  length,  as  parents  of  the  plus 
and  the  minus  lines,  were  significantly  different  from  each  other  in  every 
generation,  so  that  there  can  be  no  doubt  that  real  selections  were  made. 

TABLE  9 

Mean  spore  lengths  of  cultures  of  strain  5,  grown  prior  to  the  initiation  of  experiment  4.     Measure- 
ments in  p. 


GENERATIONS 

SPORE  LENGTHS 

RANGE  OF  MEA- 

SUREMENTS 

1 

24.4 

20-29 

2 

21.1 

17-26 

3 

22.3 

17-24 

4 

20.2 

17-24 

5 

22.2 

19-26 

6 

22.4 

19-29 

7 

20.6 

17-24 

8 

23.2 

19-29 

9 

21.9 

18-25 

10 

21.5 

17-27 

Mean 

21.98 

The  cultures  were  removed  from  Sumatra  after  three  minus  selections 
and  two  plus  selections  had  been  made.  The  fourth,  fifth,  and  sixth  selec- 
tions of  the  plus  line  show  no  decrease  in  spore  size  following  this  change 
of  climate,  since  they  are  all  at  least  equal  in  size  to  the  third  selection, 
and  the  sixth  selected  spore  equals  the  size  of  the  first  and  second  parent 
spores  of  the  line.  In  the  minus  line,  however,  the  third,  fourth,  and  fifth 
selections  show  a  decided  decrease  in  spore  size.  The  sixth  selected 
spore  is  equal  in  size  to  the  second,  but  it  should  be  noted  that  another 
chosen  spore  of  precisely  the  same  size  as  the  fifth  selected  spore  failed 
to  grow.  It  is  rather  uncertain  whether  any  of  these  changes  may  be 
attributed  to  climatic  changes. 


GENETICS  7:     Mr  1922 


174 


CARL  DOWNEY  LA  RUE 


Only  the  cultures  of  the  fifth  and  sixth  generations  were  measured. 
In  the  fifth  generation  the  culture  of  the  plus  line  had  a  greater  mean  spore 
length  than  that  of  the  minus  line,  but  in  the  next  generation  the  result 
was  reversed,  and  the  culture  of  the  minus  line  had  the  higher  mean.  The 
summation  of  the  two  generations  shows  that  the  two  lines  are  not  dis- 
tinct, the  difference  between  them  being  barely  larger  than  its  probable 
error.  The  result  of  this  experiment  is  the  same  as  that  for  the  three 
preceding  experiments,  namely,  that  selection  has  no  appreciable  result. 


22 

20 

18 

16 

14 


M 


123456 

FIGURE  10. — Graphs  showing  the  lengths  of  the  individual  spores  selected  as  parents  of 
the  plus  and  minus  lines  of  experiment  4.  P  indicates  the  plus  selections;  M,  the  minus  selec- 
tions. Lengths  in  n  are  given  as  ordinates;  the  successive  generations  as  abscissae. 

The  evidence  gained  from  two  experiments,  in  which  visible  characters 
were  selected,  is  in  entire  agreement  with  that  secured  from  the  more 
extensive  experiments  in  selection  according  to  progeny,  and  all  the  experi- 
ments, whatever  the  method  and  strain  used,  consistently  show  that 
selection  within  pure  lines  of  Pestalozzia  is  entirely  ineffective,  but  that 
rarely  mutations  occur  which  are  significantly  different  from  the  parent 
line. 

DISCUSSION 

JENNINGS  (1916,  1920)  considers  that  if  the  experiments  which  have 
been  supposed  to  demonstrate  the  negative  result  of  selection  had  dealt  with 
characters  which  were  less  likely  to  be  influenced  by  degree  of  maturity 
and  environmental  influences,  had  been  more  carefully  conducted,  and 
had  involved  a  larger  number  of  selections,  they  would  likely  have  shown  a 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  175 

positive  result.  The  Pestalozzia  investigation  has  greater  evidential 
value.  It  was  carried  on  for  10,  25,  10,  and  6  generations,  respectively, 
in  the  several  experiments.  The  characters  studied  were  such  as  to  admit 
of  certainty  as  to  maturity  of  the  observed  individuals,  and  they  were  not 
more  subject  to  environmental  influence  than  those  considered  in  investi- 
gations which  have  seemed  to  give  evidence  of  an  effect  of  selection.  In 
certain  respects,  which  have  already  been  pointed  out,  the  different  genera- 
tions have  been  more  fully  known,  and  a  larger  number  of  variates  have 
been  considered,  than  in  any  other  investigation  yet  reported.  Neverthe- 
less, the  different  experiments  consistently  fail  to  show  any  effect  of  selec- 
tion. The  evidence  secured  in  this  study  then  is  a  direct  confirmation  of 
the  results  of  JOHANNSEN  and  the  other  early  workers  on  the  pure-line 
problem.  In  attempting  an  explanation  of  the  discrepancy  between 
these  results  and  those  which  contradict  them,  it  is  necessary  to  attempt 
to  evaluate  the  experiments  that  have  shown  a  positive  selection  effect. 

TABLE  10 
Means  of  generations  measured  in  experiment  4.    Measurements  in  JJL. 


GENERATION 

PLUS-SELECTED 

LINE 

MINUS-SELECTED 
LINE 

5 

6 

21.  142  ±.110 
20.428±.114 

20.  802  +.117 
.  21.500±.112 

Means 

20.  785  +.241 

21.151±.236 

Difference  between  plus-selected  line  and  minus-selected  line = 0.366 +  0.337/J. 

The  results  of  STOCKING  (1915),  while  sufficiently  striking  and  credible, 
can  be  given  little  weight  in  a  general  consideration  since  the  abnormal 
characters  dealt  with  so  strongly  suggest  a  pathological  condition  of  the 
organism.  To  be  given  weight  in  a  discussion  of  selection  as  a  factor  in 
evolution,  the  supposed  modification  produced  by  selection  must  affect  a 
normal  character  in  a  direction  shown  by  existing  forms  in  nature  to  have 
been  actually  traversed  by  these  other  forms  in  the  course  of  their  evolu- 
tion. 

MIDDLETON  (1915)  found  selection  for  fission  rate  in  Stylonychia  effec- 
tive to  a  degree  which  must  have  been  surprising,  even  to  believers  in  the 
possibility  of  modification  by  selection.  The  great  readiness  with  which 
organisms  could  be  altered  in  MIDDLE-TON'S  experiments  indicates  that  the 
response  of  Stylonychia  to  selection  is  to  some  degree  exceptional.  ROOT 
(1918)  suggests  that  "the  inheritance  of  variations  in  fission  rate  in  Sty- 

GENETICS  7:    Mr  1922 


176  CARL  DOWNEY  LA  RUE 

lonychia  might  be  due  to  the  accumulation  of  waste  products  in  the  cyto- 
plasm of  the  slowly-dividing,  large-sized  group."  The  fact  that  MAST 
(1917)  secured  two  lines  with  different  fission  rates  mDidynium  nasutum 
without  selection  is  suggestive  that  mutation  may  offer  an  alternate 
explanation  of  MIDDLETON'S  results. 

ROOT  (1918)  working  on  Centropyxis  has  secured  data  worthy  of  consid- 
eration. In  one  experiment,  using  mass  selection,  he  found  a  decided 
effect  of  selection  on  the  number  of  spines  after  only  four  selections. 
However,  the  number  of  individuals  studied  was  very  small, — 88  in  the  high 
series  and  77  in  the  low  series.  In  another  mass  selection  the  results  were 
exactly  opposed  to  those  of  the  first;  that  is,  the  parents  with  a  low  spine 
number  produced  offspring  with  more  spines  than  those  from  parents  with 
a  large  number  of  spines.  The  latter  result  obviously  cannot  be  due  to 
selection,  though  had  it  stood  alone  it  might  have  been  so  interpreted. 
The  two  experiments  show  the  danger  of  drawing  conclusions  from  a  small 
number  of  individuals  and  a  few  selections. 

In  a  third  experiment  in  which  individuals  were  selected  according  to  the 
character  of  their  progeny  no  considerable  effect  was  gained  until  the 
fourth  selection.  From  the  fourth  selection  a  large  effect  was  secured. 
One  selected  individual  gave  offspring  all  of  which  had  a  high  spine  number. 
This  individual  (5ala  of  ROOT'S  series),  the  offspring  of  which  are  respon- 
sible for  a  decided  increase  in  the  number  of  spines  in  the  plus  series,  may 
have  been  a  mutation.  The  number  of  spines  in  the  low  series  did  not 
appreciably  decrease  during  the  selection  period.  The  whole  experiment 
included  only  a  small  number  of  individuals,  56  in  the  plus  series  and  51 
in  the  minus,  and  only  four  selections  were  made. 

In  the  fourth  experiment  in  which  mass  selection  for  number  of  spines 
was  practiced,  two  populations  were  segregated  which  varied  in  respect  to 
spine  number.  The  difference  between  the  two  populations  was  small  and 
fluctuated  from  generation  to  generation.  In  the  second  selection  period 
the  difference  was  less  than  in  the  preceding  period.  The  number  of 
individuals  observed  was  larger  than  in  the  other  experiments  but  still 
relatively  small.  It  would  have  been  interesting  to  see  what  the  result 
of  a  continuation  of  the  cultures  would  have  been.  As  it  is,  one  suspects 
the  separation  to  have  been  only  a  temporary  one,  such  as  is  shown  for 
Pestalozzia  in  table  4  of  this  paper. 

HEGNER  (1919)  carried  on  extensive  selection  experiments  on  Arcella 
and  finds  that  it  is  possible  to  separate  by  selection  within  families  groups 
which  are  distinct  in  regard  to  the  selected  character.  In  his  principal 
experiment,  however,  the  effects  of  selection  on  number  of  spines  are  by  no 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  177 

means  consistent.  The  first  set  of  selections  produced  a  higher  number  of 
spines  in  the  group  selected  for  low  spine  number  than  in  the  group  selected 
for  high  spine  number.  It  is  unfortunate  that  at  this  point  a  change  was 
made  in  the  method  of  selection.  The  author  attributes  the  effect  secured 
in  further  selections  to  the  use  of  a  better  criterion  for  the  selection.  The 
effect  may  have  been  due  to  independent  chance  fluctuations  within  the 
two  groups  and  not  to  selection  at  all  but  the  change  in  procedure  prevents 
this  being  detected.  It  would  have  been  very  interesting  to  know  whether 
or  not  such  divergences  would  have  occurred  had  the  original  method  been 
continued.  Five  further  selection  periods  showed  an  increase  in  spine 
number  in  the  group  selected  for  high  spine  number  and  a  decrease  in  the 
group  selected  for  low  number.  The  difference  between  the  high  and  the 
low  series,  however,  does  not  show  a  steady  increase,  but  fluctuates  from  one 
selection  period  to  the  next.  When  the  divergence  between  the  two  groups 
was  at  a  maximum,  selection  was  stopped,  so  that  we  do  not  know  whether 
the  divergence  would  have  decreased  or  not  had  selection  been  continued. 

On  the  cessation  of  selection  the  divergence  between  the  groups  de- 
creases very  little  for  one  period.  In  the  next  period  the  divergence  falls 
to  almost  nothing  while  in  the  next  following  period  it  again  rises  to  nearly 
half  that  secured  at  the  end  of  the  six  selection  periods.  It  is  approximate- 
ly equal  to  that  found  in  the  second  selection  period.  In  the  second  selec- 
tion period  the  increase  in  divergence  is  attributed  to  selection.  To  what 
is  it  due  in  this  non-selection  period?  It  seems  logical  to  assume  that  it 
is  due  to  the  same  cause  in  both  cases,  probably  to  the  independent 
chance  fluctuations  of  the  two  groups. 

Further  selection  for  one  period  in  the  high  line  resulted  in  a  slightly 
larger  number  of  spines  in  the  group  selected  for  low  spine  number  than 
in  that  selected  for  high  spine  number.  That  this  is  due  to  selection  is, 
of  course,  impossible. 

Three  selection  periods  were  effective  in  that  they  developed  two  diver- 
gent lines  within  the  low  line  established  by  the  early  selection.  Here 
again  the  divergence  fluctuates  from  period  to  period.  After  selection 
ceases  the  divergence  becomes  greater  than  it  was  during  the  selections. 
This  again  indicates  that  the  divergence  may  be  totally  unrelated  to  the 
selection.  In  experiment  1  of  this  paper  may  be  found  similar  divergences, 
which  were  only  temporary. 

In  another  experiment  HEGNER  practiced  selection  for  diameter  and 
spine  number  for  a  period  of  nine  days.  During  this  time  distinct  high 
and  low  lines  were  produced.  In  a  non-selection  period  of  thirteen  days 
following  the  selections  the  divergence  between  the  high  and  low  series 

GENETICS?:    Mr  1922 


178  CARL  DOWNEY  LA  RUE 

continued  to  increase  to  a  marked  degree.  Obviously  this  continued 
increase  was  not  due  to  selection;  on  the  contrary  one  would  expect  a 
lessened  divergence  between  the  high  and  low  series  following  the  cessation 
of  selection.  The  increased  divergence  during  the  non-selection  period 
was  almost  entirely  due  to  increase  in  size  and  spine  number  in  the  high 
series.  That  this  series  later  produced  some  very  large  forms,  and  that 
great  difficulty  was  experienced  in  securing  offspring  from  this  group  are 
facts  that  may  be  indicative  of  abnormality  in  the  group.  As  in  HEGNER'S 
other  experiments,  relatively  few  individuals  were  studied. 

The  extensive  and  painstaking  studies  of  JENNINGS  (1916)  on  Difflugia 
present  what  appears  to  be  the  least  questionable  evidence  of  the  effective- 
ness of  selection  yet  published.  In  one  experiment  he  made  selections  for 
seven  periods,  each  of  which,  except  the  first,  included  one  generation. 
Selection  was  made  for  number  of  spines,  individuals  with  from  1  to  3  spines 
being  chosen  as  parents  for  a  "low"  set,  and  those  with  from  5  to  8  spines 
being  retained  as  parents  of  a  "high"  set. 

The  selection  was  apparently  effective  and  is  so  interpreted  by  JEN- 
NINGS. Two  lines  were  formed  by  the  selection  which  differ  in  mean  spine 
length.  However,  a  closer  examination  shows  that  the  divergence  of  the 
two  lines  varied  from  generation  to  generation.  In  the  second  period  the 
divergence  is  equal  to  nearly  a  whole  spine  (.95  spines),  but  in  the  third 
period  the  divergence  falls  to  .03  spines,  which  means  the  lines  are  really 
identical  statistically.  From  the  third  period  on  the  divergence  continu- 
ally rises  and  falls  but  never  again  reaches  so  high  a  point  as  in  the  second 
period.  If  the  divergence  is  due  to  selection  why  is  the  effect  so  great  in 
the  second  period,  and  why  do  further  selections  result  in  a  decreased 
divergence? 

The  individual  measurements  are  not  published,  but  if  we  compute  the 
error  of  the  difference  between  the  means  of  the  two  groups,  using  the 
generation  means,  we  find  it  to  be  .  103.  The  difference  between  the  means 
of  the  two  groups  is  .22  ±  .103  spines.  The  divergence  is  barely  more  than 
twice  its  probable  error  and  so  is  not  statistically  significant. 

During  the  selection  periods  the  two  groups  showed  fluctuations  in 
regard  to  diameter  of  shell  and  length  of  spine  which  are  similar  to  those 
already  noted  for  number  of  spines.  Table  11  shows  the  divergence  of  the 
two  groups  for  each  of  the  three  characters. 

It  may  be  seen  at  a  glance  that  the  differences  do  not  steadily  increase 
with  an  increased  number  of  selections  but  rise  and  fall  as  if  by  chance. 
The  difference  in  spine  number  is  greatest  in  the  second  period,  that  of 
shell  diameter  is  greatest  in  the  fourth  period;  and  that  for  spine  length 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


179 


is  greatest  in  the  fifth  period,  falling  to  a  minus  value  in  the  sixth  period. 
The  mean  for  shell  diameter  is  the  only  one  which  is  equal  to  three  times 
its  probable  error.  Apparently  the  two  lines  are  not  demonstrably  dis- 
tinct. 

In  family  314  also,  JENNINGS  attempted  to  isolate  lines  distinct  for  spine 
number.  Selection  was  continued  for  seven  periods  in  the  same  way  as 
within  family  304.  The  means  of  the  "high"-selected  line  and  of  the 
"low"-selected  line  were  separately  computed  for  each  generation.  The 
differences  between  the  two  lines  varies  from  generation  to  generation  as 
in  family  304.  It  is  noteworthy  that  the  greatest  divergence  (1.24  spines) 
appears  in  the  first  selection  period;  the  lowest  deviation  in  the  sixth  period. 
The  difference  between  the  mean  numbers  of  spines  for  the  two  groups  is 

TABLE  11 

Differences  between  "low"  and  "high"  selected  groups  of  family 
303  of  Difflugia. 


SELECTION 

DIFFERENCES  IN 

DIFFERENCES  IN 

DIFFERENCES  IN 

PERIODS 

NUMBER  OF 

SHELL  DIAMETER  IN 

LENGTH  OF  LONGEST 

SPINES 

UNITS  OF  SCALE 

SPINES  IN  SCALE 

UNITS 

1 

.41 

.52 

.48 

2 

.95 

.63 

.57 

3 

.03 

1.16 

.39 

4 

.35 

2.88 

.02 

•  5 

.25 

.61 

.67 

6 

.34 

.95 

.29 

7 

.60 

.94 

.13 

Mean 

.22+.  103 

.93+.  26 

.76+.  299 

.42 ±.2 5  spines.  Since  the  divergence  is  less  than  twice  its  probable 
error,  we  can  only  conclude  that  the  lines  are  not  distinct  and  that  the 
apparent  difference  is  due  to  the  independent  fluctuation  of  the  two  groups. 

In  another  and  more  extensive  experiment,  JENNINGS  sought  to  isolate 
distinct  lines  by  selection  within  family  326.  Again  spine  number  was  the 
character  selected,  and  selection  was  continued  for  twelve  periods.  For 
the  first  six  periods  selection  was  ineffective  and  both  plus  and  minus  diver- 
gences between  the  "low"  and  the  "high"  groups  appeared. 

As  in  one  of  HEGNER'S  experiments,  a  change  in  the  method  of  selection, 
made  at  this  time,  prevents  us  from  knowing  what  would  have  resulted 
from  further  selections  in  which  large  numbers  of  individuals  were  used. 
Following  the  change  in  method  two  series  distinct  for  spine  number  were 


GENETICS  7:    Mr  1922 


180  CARL  DOWNEY  LA  RUE 

isolated  by  selection  during  six  periods.'  The  difference  between  the  means 
is  0.51  ±0.109  spines  or  less  than  five  times  its  probable  error. 

During  the  six  selection  periods  following  the  change  in  the  method  of 
selection  (periods  11  to  16)  the  divergence  between  the  two  groups  did 
not  constantly  increase  but  fluctuated  from  period  to  period.  In  the 
eleventh  period  it  was  .52  spines,  in  the  twelfth  it  fell  to  .49  spines,  in  the 
thirteenth  it  rose  to  .84,  the  highest  point  reached.  In  the  fourteenth 
period  the  difference  fell  to  .28  spines,  in  the  fifteenth  to  .23  spines,  which 
is  probably  not  significant,  and  in  the  sixteenth  or  last  period  it  again 
rose  to  .66  spines.  If  the  divergence  in  period  thirteen  is  due  to  selection, 
it  is  difficult  to  see  why  further  selection  for  the  same  number  of  periods 
that  caused  this  divergence,  should  cause  a  decrease  in  the  difference  of 
the  two  groups. 

After  selection  was  discontinued  the  two  groups  were  kept  under  obser- 
vation during  five  different  periods  which  varied  from  eight  to  twenty- two 
days  in  length.  The  mean  spine  number  of  the  individuals  in  the  low- 
selected  group  was  5.19  while  that  of  the  high-selected  group  was  5.58. 
The  error  of  the  difference  between  the  means  of  the  two  lines  is  0.178 
spines  while  the  difference  is  only  0.39  spines,  or  slightly  more  than  twice  its 
probable  error.  The  difference  would  not  be  considered  significant  if  it 
were  not  for  the  fact  that  the  variations  in  the  various  lines,  though  great, 
are  insufficient  to  bridge  the  gap  between  oppositely  selected  lines.  Is  it 
a  fair  criticism  that  Difflugia  is  insufficiently  plastic  to  show  rapid  changes 
from  a  certain  condition  of  the  shell  after  that  condition  is  once  attained? 
The  results  suggest  that  the  shell  itself  plays  a  part  in  determining  the 
character  of  the  new  individual  formed  by  division  of  the  old  one.  This  shell 
effect  may  be  regarded  as  superposed  upon  the  other  factors  determining 
the  form  of  the  new  shell.  Such  an  effect  might  be  expected  upon  physical 
grounds.  Naked  protoplasm  protruded  from  a  large  orifice  is  under  dif- 
ferent surface  conditions  than  if  the  orifice  were  smaller;  the  extruded 
protoplasm  would  be  expected  to  assume  a  different  form  and  size  in  the 
two  cases,  and  the  newly  secreted  shell  would  therefore  be  different.  In 
other  words,  any  accidental  size  modification  might  be  maintained  through 
several  generations,  not  through  any  protoplasmic  modification,  but 
merely  because  the  nature  of  the  shell  imposes  certain  physical  conditions 
upon  the  development  of  the  new  individuals.  A  modification  having 
once  occurred  in  the  shell  through  any  cause,  becomes  comparable  to  an 
environmental  factor  which  is  maintained  for  several  successive  genera- 
tions. The  nature  of  new  individuals  is  largely  determined  by  the  environ- 
ment, and  of  the  environmental  factors  the  shell  is  one  of  the  chief.  If  this 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  181 

suggestion  has  any  validity,  it  follows  that  the  two  groups,  while  apparently 
different,  were  not  significantly  different,  even  while  selection  was  being 
practiced. 

If  two  lines  are  isolated  within  a  strain  and  cultivated  separately  these 
lines  will  be  independently  subjected  to  the  environmental  influences 
which  cause  variation  for  the  character  studied.  The  lines  will  tend  to 
coincide  if  they  are  subjected  to  these  influences  at  the  same  time  and  to 
the  same  degree.  This  is  the  case  with  the  strain  of  Festal ozzia  studied  in 
the  author's  experiment  2  and  is  shown  in  figure  7.  It  is  apparent  that 
there  is  a  certain  rhythm,  either  in  the  organism  itself  or  in  the  environ- 
mental factors,  which  causes  upward  and  downward  swings  of  the  organism 
in  respect  to  the  character  under  observation.  Since  great  care  was  taken 
to  have  the  two  groups  subject  to  identical  conditions  at  all  times  the  two 
groups  follow  one  another  very  closely  in  these  swings.  In  experiment  1 
the  swings  follow  each  other  less  closely  but  with  some  exceptions  there  is  a 
strong  similarity  of  behavior  in  the  two  lines  as  is  shown  in  figure  5.  In 
MAST'S  work  with  Didynium,  where  two  groups  have  been  separated, 
apparently  by  a  mutation,  these  groups  fluctuate  in  the  same  direction 
at  the  same  time,  with  remarkable  unison. 

If  two  lines  are  isolated  and  cultivated  separately,  but  the  offspring  are 
produced  in  such  a  way  that  those  of  the  two  lines  are  subjected  to  the 
environmental  factors  causing  variation  for  a  given  character,  at  a  different 
time,  or  in  a  different  degree,  each  of  the  lines  will  fluctuate  in  a  different 
way,  and  this  will  cause  divergence  between  the  two.  However  if  the 
lines  are  continued  the  fluctuations  will  compensate  one  another  so  that 
in  the  end  the  two  lines  will  be  found  to  give  practically  the  same  mean 
value  for  the  character  studied. 

To  the  writer  it  appears  that  JENNINGS'S  results  with  Difflugia,  ROOT'S 
with  Centropyxis,  and  HEGNER'S  with  Arcella,  are  explained  by  the  above 
assumption  quite  as  well  as  are  his  own  with  Pestalozzia.  If  this  be  true 
they  tend  rather  to  demonstrate  the  ineffectiveness  of  selection  within 
pure  lines  than  the  opposite. 

It  is  warranted  to  express  the  belief  that  the  "pure-line"  hypothesis  is 
still  valid.  The  writer  does  not  wish  to  express  an  opinion  that  ultimately 
the  effectiveness  of  selection  may  not  be  shown,  but  he  feels  that  the  bur- 
den of  proof  lies  with  those  who  question  the  results  of  JOHANNSEN  and 
his  followers.  At  the  present  time  incontestable  evidence  of  an  effect  of 
selection  is  wanting.  From,  the  recent  investigations  made  in  this  field 
it  is  obvious  that  the  situation  is  vastly  more  complicated  than  was 
formerly  supposed.  The  truth  or  falsity  of  the  "pure-line"  hypothesis 

GENETICS  7:    Mr  1922 


182  CARL  DOWNEY  LA  RUE 

will  be  finally  determined  by  numerous  investigations  with  various  or- 
ganisms, involving  infinite  care  and  toil.  There  is  need  of  more  investiga- 
tions of  the  type  which  JENNINGS  has  made  on  Paramecium  and  Difflugia, 
but  carried  for  longer  periods,  with  organisms  more  favorable  for  study, 
if  they  may  be  found. 

Nothing  has  been  said  in  this  paper,  of  the  bearing  of  the  "pure-line" 
hypothesis  on  theories  of  evolution.  Almost  every  worker  who  has  made 
careful  biometric  studies  of  highly  variable  groups  has  recorded  the  occur- 
rence of  mutations  or  "something  like  mutations."  JENNINGS,  ROOT, 
MAST,  and  HEGNER  have  all  recorded  them  for  the  lower  animals.  BAR- 
BER (1907)  noted  the  occurrence  of  mutation  in  yeasts;  STEVENS  (1920) 
has  reported  a  number  of  mutations  in  Helminthosporium ;  and  the  writer 
has  found  mutations  in  Pestalozzia.  It  is  unnecessary  to  catalogue  the 
numerous  cases  of  mutation  recorded  in  various  groups  of  higher  plants 
and  animals.  Further  careful  study  will  doubtless  reveal  many  more. 
When  more  is  known  of  their  occurrence,  frequency,  and  reaction  to 
environment,  to  other  existing  organisms,  and  to  one  another,  may  we  not 
be  able  to  conceive  of  evolution,  even  without  cumulative  growth  of  char- 
acters by  the  selection  of  fluctuating  variations? 

CONCLUSIONS 

Selections  according  to  progeny  within  pure  strains  of  Pestalozzia  were 
made  for  length  of  spores  for  ten  generations,  and  for  length  of  spore 
appendages  for  twenty-five  generations,  without  result.  Selections  ac- 
cording to  visible  characters  were  made  for  spore  length  for  ten  genera- 
tions in  one  experiment  and  for  six  generations  in  another,  without 
establishing  differences  between  the  plus-selected  and  minus-selected  lines. 
Whatever  method  and  strain  may  be  employed,  selection  is  totally  in- 
effective in  establishing  distinct  lines  within  pure  strains  of  Pestalozzia 
Guepini. 

Mutations  which  give  rise  to  lines  significantly  different  from  the  parent 
lines,  infrequently  occur. 

LITERATURE  CITED 

BARBER,  M.A.,  1907     On  heredity  in  certain  micro-organisms.  Kansas  Univ.  Sci.  Bull.  4, 48  pp. 
EWING,  H.  E.  1914  a  Pure  line  inheritance  and  parthenogenesis,  Biol.  Bull.  26:  25-35. 
1914  b  Notes  on  regression  in  a  pure  line  of  plant  lice.     Biol.  Bull.  27:  164-168. 
1916    Eighty-seven  generations  in  a  parthenogenetic  pure  line  of  Aphis  avenae  Fab.  Biol. 

Bull.  31:53-112. 

HANEL,  ELISE,  1908  Vererbung  bei  ungeschlectlicher  Fortpflanzung  von  Hydra  grisea.  Jenaische 
Zeitschr.  43:221-372. 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA  183 

HARRIS,  J.  A.,  1911    The  biometric  proof  of  the  pure  line  theory.    Amer.  Nat.  45:346-363. 
HEGNER,  R.  W.,  1919    Heredity,  variation,  and  the  appearance  of  diversities  during  the  vege- 
tative reproduction  of  Arcella  dentata.     Genetics  4: 95-150. 
JENNINGS,  H.  S.,   1908    Heredity,  variation  and  evolution  in  Protozoa.     II.  Heredity  and 

variation  in  size  and  form  in  Paramecium  with  studies  of  growth,  environmental  action 

and  selection.     Proc.  Amer.  Philos.  Soc.  47:393-546. 
1916    Heredity,  variation  and  the  results  of  selection  in  the  uniparental  reproduction  of 

Difflugia  corona.     Genetics  1 :  407-534. 
1920    Life  and  death,  heredity  and  evolution  in  unicellular  organisms.     233  pp.  Boston: 

Richard  G.  Badger. 
JOHANNSEN,  W.,  1913    Elemente  der  exakten  Erblichkeitslehre.    Zweite  Ausgabe,  xi-f-723  pp. 

Jena:  Gustav  Fischer. 
KAUITMAN,  C.  H.,  1908    A  contribution  to  the  physiology  of  the  Saprolegniaceae,  with  special 

reference  to  the  variations  of  the  sexual  organs.     Annals  of  Bot.  87: 361-386. 
LA  RUE,  CARL  D.,  1920    Isolating  single  spores.    Bot.  Gaz.  70: 317-318. 
LA  RUE,  CARL  D.,  and  BARTLETT,  H.  H.,  1922    A  demonstration  of  numerous  distinct  strains 

within  the  nominal  species  Pestalozzia  Guepini  Desm.    Amer.  Jour.  Bot.  9:79-92. 
LASHLEY,  K.  S.,  1915    Inheritance  in  the  asexual  reproduction  of  Hydra.    Jour.  Exper.  Zool. 

19:  157-210. 

1916    Inheritance  in  the  asexual  reproduction  of  Hydra.    Jour.  Exp.  Zool.  20: 19-26. 
MAST,  S.  O.,  1917    Mutation  in  Didynium  nasutum.    Amer.  Nat.  51:  351-360. 
MIDDLETON,  A.  R.  1915     Heritable  variations  and  the  results  of  selection  in  the  fission  rate  of 

Stylonychia  pustulata.    Jour.  Exper.  Zool.  19:451-503. 
MORGAN,  T.  H.,  1916    A  critique  of  the  theory  of  evolution,    x-f  197  pp.  Princeton:  Princeton 

University  Press. 
PEARSON,  KARL,  1910    Darwinism,  biometry  and  some  recent  biology.    I.    Biometrika  7: 

368-385. 
1914    Tables  for  statisticians  and  biometricians.     Ixxxiii  +  143  pp.  Cambridge:  Cambridge 

University  Press. 

PIETERS,  A.  J.,  1915    New  species  of  Achlya  and  of  Saprolegnia.    Bot.  Gaz.  60:  483-490. 
ROOT,  F.  M.,  1918    Inheritance  in  the  asexual  reproduction  of  Centropyxis  aculeata.     Genetics 

3:  173-206. 
STEVENS,  F.  L.,  1920    Heterogenetic  saltation  in  the  genus  Helminthosporium,  with  comments 

on  morphology  and  culture  characters.      Paper  read  before  the  Bot.  Soc.  Amer.,  Myco- 

logical  Section,  Chicago,  Dec.  1920. 
STOCKING,  RUTH  J.,  1915    Variation  and  inheritance  of  abnormalities  occurring  after  conjugation 

in  Paramecium  caudatum.    Jour.  Exper.  Zool.  19 :  387-449. 
WOLLENWEBER,  H.  W.,   1914    Identification  of  species  of  Fusarium  occurring  on  the  sweet 

potato,  Ipomoea  Batatas.     Jour.  Agric.  Res.  2:  251-285. 


GENETICS  7:    Mr  1922 


184 


CARL  DOWNEY  LA  RUE 


APPENDIX 

Tables  of  constants  of  experiments  1  and  2 

TABLE  12 

Means  and  standard  deviations  of  spore  lengths  of  cultures  grown  in  experiment  1.  The  mean  of 
each  culture  was  calculated  from  at  least  100  measurements.  Values  are  given  in  units  of  the  scale  of 
the  eye-piece  micrometer  used.  1  unit  =  1.8  microns.  Single  spore  culture  of  parent  strain  (No.  29} . 
Mean  =  13.80,  ff  =  1.58. 

Generation  1  of  experiment 


CULTURE 

MEAN 

a 

CULTURE 

MEAN 

9 

1 

14.46 

1.55 

11 

14.16 

1.43 

2 

13.90 

1.76 

12 

13.72 

1.82 

3 

13.61 

1.85 

13 

13.64 

1.43 

4 

13.97 

1.63 

14 

13.62 

1.54 

5 

13.51 

1.79 

15 

14.18 

1.84 

6 

14.02 

1.90 

16 

13.58 

1.67 

7 

13.71 

1.80 

17 

13.85 

1.87 

8 

13.57 

1.71 

18 

13.79 

1.82 

9 

13.95 

1.47 

19 

13.95 

1.65 

10 

13.71 

1.71 

20 

13.89 

1.52 

Mean  of  generation  13.84. 


Generation  2 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

a 

Mean 

<T 

Mean 

0- 

1 

14.03 

1.41 

14.23 

1.66 

13.40 

1.10 

2 

14.29 

1.44 

13.99 

1.38 

3 

14.94 

1.52 

14.19 

.39 

4 

13.95 

1.43 

14.00 

.29 

5 

14.33 

1.49 

13.82 

.39 

6 

14.35 

1.52 

14.00 

.27 

7 

14.94 

1.55 

14.31 

.43 

8 

13.43 

1.33 

14.12 

.52 

9 

13.72 

1.54 

14.17 

1.62 

10 

13.94 

1.93 

14.15 

1.33 

Generation 

means 

14.19 

14.10 

13.40 

SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


185 


TABLE  12  (continued) 
Generation  3 


PLUS  SEL 

ECTIONS 

MINUS  SE] 

.ECTIONS 

INTERMEDIA! 

'E  GROUP 

Mean 

a 

Mean 

<r 

Mean 

<r 

1 

14.46 

1.26 

13.55 

.24 

14.77 

1.38 

2 

13.88 

1.28 

13.92 

.22 

3 

14.19 

1.21 

13.60 

.38 

4 

14.11 

1.09 

13.61 

.29 

5 

14.34 

1.25 

13.60 

.96 

6 

13.75 

1.18 

13.54 

.18 

7 

13.75 

1.16 

13.79 

.33 

8 

14.22 

1.33 

13.77 

.21 

9 

13.74 

1.07 

13.59 

.33 

10 

13.88 

1.15 

13.70 

.31 

Generation 

means 

14.03 

13.67 

14.77 

Generation  4 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

<r 

Mean 

<r 

1 

13.50 

1.17 

13.27 

1.13 

13.55 

1.14 

•       2 

13.62 

1.22 

13.68 

1.24 

3 

13.87 

1.04 

13.63 

1.32 

4 

13.11 

1.12 

13.79 

1.13 

5 

13.37 

1.11 

13.32 

0.99 

6 

13.93 

1.33 

13.09 

1.29 

7 

13.78 

1.12 

13.64 

1.07 

8 

13.53 

1.37 

13.44 

1.29 

9 

13.27 

1.01 

13.55 

0.95 

10 

13.59 

1.21 

13.52 

1.07 

Generation 

means 

13.56 

13.49 

13.55 

GENETICS  7:     Mr  1922 


186 


CARL  DOWNEY  LA  RUE 


TABLE  12  (continued) 
Generation  5 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

a 

1 

13.92 

1.17 

13.93 

1.12 

14.03 

1.32 

2 

13.55 

1.04 

14.19 

1.26 

3 

14.07 

1.31 

13.43 

.29 

4 

13.60 

1.24 

13.75 

.13 

5 

13.77 

1.02 

13.41 

.21 

6 

13.88 

1.10 

13.71 

.42 

7 

13.40 

1.19 

13.81 

.10 

8 

13.47 

1.37 

14.14 

.27 

9 

14.05 

1.02 

13.53 

.12 

10 

13.63 

1.10 

14.04 

.19 

Generation 

means 

13.73 

13.79 

14.03 

Generation  6 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

a 

Mean 

a 

Mean 

a 

1 

13.73 

1.17 

13.75 

1.18 

13.31 

1.12 

2 

13.74 

.18 

13.65 

1.19 

. 

3 

13.91 

.12 

13.75 

1.33 

4 

14.14 

.01 

13.60 

0.88 

5 

13.62 

.05 

13.23 

1.08 

6 

13.65 

.00 

13.87 

1.06 

7 

13.64 

.11 

13.68 

1.18 

8 

13.69 

.10 

14.10 

1.13 

9 

13.84 

.20 

13.66 

1.36 

10 

13.50 

1.15 

13.91 

1.24 

Generation 

means 

13.75 

13.72 

13.31 

SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


187 


TABLE  12  (continued) 
Generation  7 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

a 

Mean 

<r 

Mean 

<r 

1 

13.84 

1.28 

13.26 

1.06 

2 

13.76 

1.21 

3 

13.59 

1.13 

14.17 

1.40 

4 

13.78 

1.09 

5 

13.33 

1.33 

6 

14.08 

0.98 

7 

13.86 

1.12 

13.76 

1.33 

8 

13.45 

0.97 

13.67 

1.37 

9 

13.87 

1.22 

13.17 

1.66 

10 

13.75 

1.22 

Generation 

means 

13.73 

13.69 

13.26 

Generation  8 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

<r 

1 

13.46 

0.95 

13.47 

0.98 

2 

13.65 

1.11 

3 

13.79 

1.17 

13.03 

1.01 

4 

13.93 

1.06 

13.79 

1.05 

5 

13.89 

1.07 

13.49 

0.98 

6 

13.18 

1.11 

13.60 

1.15 

7 

13.42 

1.05 

13.59 

1.13 

8 

13.80 

1.14 

13.17 

1.08 

9 

13.47 

1.08 

10 

13.49 

1.15 

Generation 

' 

means 

13.61 

13.45 

13.47 

GENETICS  7:    Mr  1922 


188 


CARL  DOWNEY  LA  RUE 


TABLE  12  (continued) 
Generation  9 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

<r 

Mean 

<r 

1 

13.46 

.23 

13.43 

1.07 

13.29 

1.03 

2 

13.50 

.17 

13.56 

1.21 

3 

13.28 

.08 

13.92 

1.03 

4 

13.71 

.13 

13.84 

1.02 

5 

13.26 

.06 

13.57 

0.99 

6 

13.53 

.09 

13.21 

1.34 

7 

13.55 

.31 

13.47 

1.11 

8 

13.45 

1.04 

13.68 

1.05 

9 

13.38 

1.17 

13.25 

0.98 

10 

13.31 

1.08 

13.75 

1.08 

Generation 

means 

13.44 

13.57 

13.29 

Generation  10 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

<r 

1 

13.45 

1.22 

13.05 

1.03 

13.35 

1.03 

2 

13.31 

1.15 

13.13 

1.38 

3 

13.89 

1.33 

13.14 

1.00 

4 

13.03 

0.95 

12.96 

1.02 

5 

13.71 

0.94 

13.27 

0.91 

6 

13.86 

0.96 

13.26 

0.88 

7 

13.67 

0.99 

13.19 

1.18 

8 

13.12 

1.08 

9 

13.57 

1.11 

12.92 

1.17 

10 

13.54 

1.04 

13.44 

1.07 

Generation 

means 

13.56 

13.15 

13.35 

Means  of  all  cultures  in  experiment  1 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

13.736+0.0311 
<r=0.435±0.0219 

13.  679  ±0.0226 
<r=0.331±0.0159 

13.640  +  0.070 
o-=0.326±0.0492 

Difference  between  plus  selections  and  minus  selections =0.05  7 +  0.03  8 
Difference  between  plus  selections  and  intermediate  group  =0.10  +  0.058 
Difference  between  minus  selections  and  intermediate  group  =0.039  +  0.054. 


SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


189 


TABLE  13 

Means  and  standard  deviations  of  appendage  lengths  of  cultures  studied  in  experiment  2.  The 
mean  of  each  culture  was  calculated  from  100  or  more  measurements.  Values  are  given  in  units  of 
micrometer  scale:  1  unit  =  1.8  microns.  Single-spore  culture  of  parent  strain  (No.  29).  Mean  — 
12.42,  a =2. 08. 

Generation  1  of  experiment 


CULTURE 

MEAN 

a 

CULTURE 

MEAN 

0- 

1 

12.67 

2.20 

11 

13.62 

2.21 

2 

12.50 

2.22 

12 

13.52 

3.07 

3 

13.58 

2.30 

13   . 

13.69 

2.96 

4 

13.57 

2.40 

14 

13.48 

2.55 

5 

13.85 

2.43 

15 

12.48 

2.54 

6 

13.46 

2.26 

16 

14.01 

2.52 

7 

13.64 

2.52 

17 

12.01 

2.81 

8 

13.10 

2.30 

18 

13.08 

2.42 

9 

13.66 

2.34 

19 

13.74 

2.72 

10 

13.81 

2.29 

20 

12.46 

2.33 

Mean  of  generation  13.30 


Generation  2 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

<r 

Mean 

<r 

1 

11.70 

2.00 

11.59 

2.17 

12.80 

2.28 

2 

12.16 

1.08 

11.42 

2.24 

3 

12.09 

1.95 

11.60 

2.31 

4 

12.03 

1.88 

11.44 

2.07 

5 

11.92 

2.26 

11.40 

2.06 

6 

11.44 

2.19 

10.63 

1.91 

7 

11.49 

2.03 

11.36 

2.22 

8 

11.92 

2.71 

11.60 

2.13 

9 

11.14 

2.16 

11.68 

2.29 

10 

11.97 

1.98 

11.42 

2.03 

Generation 

means 

11.79 

11.41 

12.80 

GENETICS  7:    Mr  1922 


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CARL  DOWNEY  LA  RUE 


TABLE  13  (continued) 
Generation  3 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

CULTURE 

Mean 

<r 

Mean 

a 

Mean 

a 

1 

10.05 

1.77 

10.31 

2.34 

11.55 

2.33 

2 

9.99 

2.20 

10.41 

1.67 

3 

9.84 

2.07 

10.44 

2.20 

4 

11.29 

2.17 

11.01 

2.09 

5 

10.64 

2.16 

10.78 

2.19 

6 

10.85 

2.08 

10.17 

1.60 

7 

10.48 

2.12 

10.01 

2.04 

8 

10.53 

2.07 

10.11 

1.76 

9 

10.75 

1.96 

10.49 

2.27 

10 

11.52 

1.99 

10.41 

2.10 

Generation 

means 

10.59 

10.41 

11.55 

Generation  4 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

<T 

Mean 

o- 

1 

10.60 

1.88 

12.42 

2.08 

11.60 

2.08 

2 

9.99 

2.16 

11.69 

2.60 

3 

11.04 

1.83 

10.97 

1.98 

4 

10.61 

2.18 

11.32 

1.83 

5 

10.18 

1.94 

11.78 

2.36 

6 

11.48 

2.18 

11.23 

2.43 

7 

12.14 

2.17 

10.99 

2.03 

8 

11.40 

2.20 

11.72 

2.27 

9 

11.18 

1.77 

11.82 

2.36 

10 

10.55 

1.93 

11.22 

2.17 

Generation 

means 

10.92 

11.52 

11.60 

SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


191 


TABLE  13  (continued) 
Generation  5 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

a 

Mean 

a 

Mean 

<T 

1 

11.82 

2.46 

11.93 

1.96 

11.55 

2.43 

2 

12.22 

2.08 

10.95 

2.44 

3 

11.60 

1/98 

10.51 

2.31 

4 

12.42 

2.38 

11.13 

2.67 

5 

11.64 

2.17 

10.54 

1.96 

6 

12.33 

2.12 

11.52 

2.14 

7 

12.39 

2.22 

11.17 

2.35 

8 

12.19 

2.30 

10.26 

2.46 

9 

12.04 

2.43 

11.15 

2.28 

10 

12.33 

2.63 

10.45 

2.37 

Generation 

means 

12.10 

10.96 

11.55 

Generation  6 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

a 

Mean 

a 

Mean 

a 

1 

10.96 

2.57 

11.79 

2.36 

12.57 

2.34 

2 

11.92 

2.36 

11.47 

2.31 

3 

11.91 

2.13 

11.39 

2.30 

4 

11.04 

2.31 

12.40 

2.21 

5 

11.60 

2.16 

12.00 

2.37 

6 

11.50 

1.90 

11.08 

2.18 

7 

11.80 

2.60 

11.88 

2.41 

8 

11.58 

1.86 

11.82 

2.47 

9 

11.19 

2.61 

11.20 

2.31 

10 

11.93 

2.61 

12.02 

2.84 

Generation 

means 

11.54 

11.71 

12.57 

GENETICS  7:     Mr  1922 


192 


CARL  DOWNEY  LA  RUE 


TABLE  13  (continued) 
Generation  7 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

CULTURE 

Mean 

<r 

Mean 

r 

Mean 

o- 

1 

12.11 

2.16 

11.86 

2.34 

12.45 

2.30 

2 

12.34 

1.98 

3 

12.08 

2.25 

12.29 

2.58 

4 

12.10 

2.27 

12.36 

2.22 

5 

12.49 

2.74 

6 

12.16 

2.35 

12.14 

2.15 

7 

11.87 

2.31 

11.94 

2.25 

8 

12.38 

1.86 

12.35 

2.44 

9 

12.25 

2.13 

12.25 

2.10 

10 

12.30 

1.65 

11.82 

2.20 

Generation 

means 

12.18 

12.16 

12.45 

Generation  8 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

tr 

Mean 

<r 

1 

11.58 

1.99 

11.84 

2.09 

11.65 

2.18 

2 

11.72 

1.96 

11.91 

2.20 

3 

12.47 

1.94 

10.68 

1.74 

4 

11.41 

2.26 

11.92 

2.05 

5 

11.42 

2.12 

12.08 

2.14 

6 

11.71 

2.17 

12.03 

2.09 

7 

11.51 

2.22 

12.34 

2.02 

8 

11.39 

2.24 

11.23 

2.19 

9 

11.95 

2.15 

12.04 

2.33 

10 

10.81 

2.18 

11.87 

2.30 

Generation 

means 

11.60 

11.79 

11.65 

SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


193 


TABLE  13  (continued) 
Generation  9 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

CULTURE 

Mean. 

a 

Mean 

a 

Mean 

a 

1 

10.45 

1.98 

10.40 

1.92 

10.46 

2.13 

2 

10.88 

2.16 

10.83 

1.94 

3 

10.66 

2.12 

9.47 

2.04 

4 

10.21 

2.03 

9.37 

1.86 

5 

10.27 

1.94 

9.75 

1.84> 

6 

10.05 

2.00 

10.30 

2.06 

7 

10.59 

2.23 

9.85 

2.15 

8 

9.70 

2.02 

10.13 

2.17 

9 

9.95 

1.74 

10.32 

2.02 

10 

10.63 

2.16 

10.81 

1.93 

Generation 

means 

10.34 

10.12 

10.46 

Generation  10 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<T 

Mean 

<r 

Mean 

<T 

1 

9.07- 

1.86 

10.19 

2.07 

9.47 

1.55 

2 

9.49 

1.79 

9.64 

2.20 

3 

9.27 

1.98 

10.03 

2.14 

4 

10.20 

1.80 

9.82 

1.84 

5 

9.13 

1.69 

10.00 

1.60 

6 

9.88 

2.07 

9.87 

2.03 

7 

9.54 

1.68 

9.98 

1.93 

8 

10.22 

1.92 

9.79 

1.69 

9 

9.84 

1.99 

10 

9.88 

1.94 

10.18 

1.97 

Generation 

means 

9.63 

9.93 

9.47 

GENETICS  7:    Mr  1922 


194 


CARL  DOWNEY  LA  RUE 


TABLE  13  (continued) 
Generation  11 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

a 

Mean 

9 

Mean 

0- 

1 

10.82 

2.09 

12.60 

1.93 

2 

12.30 

2.25 

3 

12.07 

2.20 

4 

11.32 

2.14 

5 

11.33 

2.17 

6 

12.23 

2.19 

7 

11.99 

2.07 

8 

11.75 

2.10 

9 

11.46 

2.02 

10 

10.95 

1.83 

11.87 

2.13 

Generation 

means 

11.62 

11.87 

12.60 

Generation  12 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

<r 

1 

11.22 

2.09 

11.40 

2.08 

11.19 

1.95 

2 

11.07 

1.96 

10.33 

2.11 

3 

4 

10.43 

2.05 

10.94 

2.27 

5 

10.76 

2.12 

10.24 

1.78 

6 

10.63 

2.09 

10.92 

2.22 

7 

8 

10.77 

2.17 

10.91 

2.14 

9 

10 

10.69 

1.94 

10.22 

2.01 

Generation 

means 

10.79 

10.71 

11.19 

SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


195 


TABLE  13  (continued) 
Generation  13 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

CULTURE 

Mean 

a 

Mean 

<r 

Mean 

<r 

1 

10.53 

2.04 

9.77 

1.76 

10.37 

2.05 

2 

10.07 

1.87 

10.49 

2.16 

3 

11.22 

2.08 

10.75 

2.38 

4 

10.61 

2.33 

10.65 

1.69 

5 

9.76 

2.09 

10.72 

2.32 

6 

10.52 

2.20 

10.04 

1.99 

7 

10.94 

2.33 

10.87 

2.18 

8 

10.66 

1.93 

10.94 

2.19 

9 

10.35 

2.31 

10.19 

2.26 

10 

10.83 

2.25 

9.63 

2.32 

Generation 

means 

10.55 

10.41 

10.37 

Generation  14 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

a 

Mean 

<r 

Mean 

a 

1 

11.48 

1.92 

10.58 

2.15 

10.33 

2.10 

2 

10.47 

1.76 

12.16 

2.17 

3 

10.64 

1.82 

4 

10.74 

1.83 

10.33 

2.13 

5 

11.98 

2.55 

14.85* 

2.70 

6 

12.52 

2.33 

7 

11.71 

2.11 

11.24 

2.35 

8 

12.29 

2.16 

9 

11.29 

2.10 

12.30 

1.98 

10 

11.68 

2.22 

11.76 

2.22 

Generation 

means 

11.48 

11.90 

10.33 

*Mutant. 


GENETICS  7:    Mr  1922 


196 


CARL  DOWNEY  LA  RUE 


TABLE  13  (continued) 
Generation  15 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

<r 

Mean 

a 

1 

11.67 

2.11 

11.78 

2.08 

11.80 

2.46 

2 

11.78 

2.07 

11.47 

1.76 

3 

11.90 

1.87 

12.12 

2.11 

4 

11.34 

2.34 

11.65 

1.91 

5 

11.75 

2.04 

11.36 

2.02 

6 

11.33 

2.33 

11.07 

1.79 

7 

11.35 

2.28 

11.20 

1.98 

8 

12.50 

2.24 

11.71 

2.06 

9 

11.27 

2.21 

11.77 

2.04 

10 

11.22 

1.85 

10.47 

2.19 

Generation 

means 

11.61 

11.46 

11.80 

Generation  16 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

ff 

1 

11.37 

2.08 

10.86 

2.05 

11.00 

2.45 

2 

11.52 

2.10 

10.41 

2.09 

3 

10.50 

2.07 

10.79 

2.21 

4 

11.57 

2.01 

10.82 

2.26 

5 

11.27 

2.07 

10.55 

1.97 

6 

11.49 

1.98 

7 

11.17 

2.33 

11.25 

1.96 

8 

11.76 

2.25 

10.73 

1.73 

9 

12.09 

1.78 

10.22 

1.65 

10 

11.77 

2.04 

Generation 

means 

11.45 

10.70 

11.00 

SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


197 


TABLE  13  (continued) 
Generation  17 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<T 

Mean 

<r 

Mean 

ff 

1 

10.70 

1.98 

9.97 

1.63 

10.34 

1.81 

2 

9.83 

1.82 

10.60 

1.93 

3 

10.36 

1.77 

9.92 

1.87 

4 

10.46 

2.01 

10.09 

1.75 

5 

10.61 

1.92 

9.88 

1.80 

6 

10.32 

2.16 

10.37 

1.87 

7 

10.17 

1.72 

10.01 

1.73 

. 

8 

10.87 

1.67 

10.17 

2.00 

9 

10.01 

1.83 

10.60 

2.21 

10 

9.80 

1.96 

10.29 

1.86 

Generation 

means 

10.31 

10.19 

10.34 

Generation  18 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

CULTURE 

Mean 

<r 

Mean 

<r 

Mean 

<r 

1 

9.56 

1.97 

10.80 

1.95 

2 

9.94 

2.43 

10.66 

2.02 

3 

10.47 

1.82 

9.92 

1.54 

4 

10.36 

2.01 

5 

10.00 

1.78 

6 

9.72 

2.08 

10.45 

1.74 

7 

10.46 

1.93 

8 

9 

10.02 

2.16 

10.26 

1.74 

10 

9.85 

2.04 

10.62 

1.79 

Generation 

means 

9.99 

10.39 

10.80 

GENETICS  7:    Mr  1922 


198 


CARL  DOWNEY  LA  RUE 


TABLE  13  (continued) 


Generation  19 


PLUS  SELECTIONS 

MINUS  SELECTIONS. 

INTERMEDIATE  GROUP 

Mean 

<7 

Mean 

9 

Mean 

<r 

1 

11.60 

1.74 

11.37 

1.89 

2 

11.49 

2.08 

11.41 

1.97 

3 

11.12 

2.02 

11.22 

1.97 

4 

11.14 

2.16 

10.85 

1.99 

5 

11.12 

2.18 

10.72 

1.69 

6 

11.49 

2.07 

11.71 

2.11 

7 

11.69 

1.96 

11.91 

2.28 

8 

11.61 

1.98 

12.30 

2.04 

9 

11.44 

1.99 

12.15 

1.98 

10 

11.79 

2.18 

Generation 

means 

11.49 

11.57 

11.37 

Generation  20 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

<r 

1 

11.59 

2.05 

11.35 

1.91 

10.79 

1.81 

2 

10.89 

2.05 

11.11 

1.86 

3 

11.00 

2.11 

11.05 

1.93 

4 

10.55 

1.93 

11.14 

1.94 

5 

10.69 

2.00 

10.68 

2.18 

6 

11.22 

1.90 

11.41 

1.96 

7 

10.68 

1.91 

10.88 

1.78 

8 

10.98 

2.14 

10.95 

1.81 

9 

10.53 

2.02 

10.02 

2.03 

10 

10.86 

2.07 

10.49 

2.10 

Generation 

means 

10.90 

10.90 

10.79 

SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


199 


TABLE  13  (continued) 
Generation  21 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

a 

1 

11.70 

2.26 

11.43 

2.01 

10.36 

2.01 

2 

11.47 

1.96 

11.01 

1.87 

3 

10.49 

1.92 

9.64 

1.70 

4 

10.74 

1.69 

5 

10.80 

2.09 

10.86 

2.00 

6 

11.37 

1.94 

7 

10.78 

1.98 

10.88 

2.10 

8 

8.99 

1.87 

11.44 

1.99 

9 

11.95 

2.09 

10.46 

2.02 

10 

12.21 

2.04 

10.59 

1.84 

Generation 

means 

11.13 

10.85 

10.36 

Generation  22 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

o- 

1 

11.76 

2.07 

11.96 

2.00 

10.58 

1.94 

2 

11.22 

2.10 

10.85 

1.99 

3 

11.14 

2.00 

11.39 

2.33 

4 

11.06 

1.74 

10.75 

2.26 

5 

10.66 

1.74 

11.21 

1.74 

6 

11.11 

1.95 

11.76 

2.32 

7 

11.37 

2.15 

12.16 

2.20 

8 

11.42 

2.14 

11.56 

1.67 

9 

11.38 

2.14 

11.30 

1.87 

10 

11.04 

1.83 

11.80 

2.30 

Generation 

means 

11.22 

11.47 

10.58 

GENETICS  7:    Mr  1922 


200 


CARL  DOWNEY  LA  RUE 


TABLE  13  (continued) 
Generation  23 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

(7 

Mean 

(T 

1 

11.37 

2.02 

10.89 

1.95 

12.13 

2.28 

2 

10.80 

.92 

11.29 

1.84 

3 

11.29 

.77 

10.96- 

1.80 

4 

11.50 

.80 

10.82 

1.81 

5 

10.94 

.95 

11.50 

1.96 

6 

11.30 

.83 

11.53 

1.77 

7 

11.44 

2.05 

8 

10.98 

1.90 

11.28 

1.86 

9 

10.98 

1.91 

11.56 

1.88 

10 

10.87 

1.82 

11.46 

1.99 

Generation 

means 

11.11 

11.27 

12.13 

Generation  24 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<T 

Mean 

cr 

Mean 

a 

1 

11.48 

2.26 

11.48 

1.92 

11.70 

2.52 

2 

11.56 

2.23 

10.88 

2.25 

3 

10.40 

1.89 

4 

11.35 

1.98 

5 

10.63 

2.02 

6 

11.26 

2.22 

7 

11.11 

2.00 

8 

11.22 

1.93 

9 

11.37 

2.14 

10 

11.46 

2.17 

Generation 

means 

11.52 

11.12 

11.70 

SELECTION  WITHIN  PURE  LINES  OF  PESTALOZZIA 


201 


TABLE  13  (continued) 
Generation  25 


CULTURE 

PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

Mean 

<r 

Mean 

a 

Mean 

a 

1 

2 

10.33 
10.45 

1.62 
2.00 

10.90 

1.91 

3 

11.45 

2.23 

4 

10.44 

1.91 

5 

10.58 

2.17 

10.46 

1.96 

6 

10.91 

1.87 

7 

10.72 

1.85 

8 

10.87 

2.31 

9 

10 

Generation 

means 

10.73 

10.68 

10.90 

Means  of  all  cultures  in  experiment  2 


PLUS  SELECTIONS 

MINUS  SELECTIONS 

INTERMEDIATE  GROUP 

11.330  +  0.469 
<r  =  l.  06  +  0.  033 

11.235  +  0.043 
<7=0.97±0.030 

11.39  +  0.126 
<r=0.  034  +  0.  089 

Difference  between  plus  and  minus  selections =0.095 +  0.064 

Difference  between  plus  selection  and  intermediate  group  =0.060+0.134 

Difference  between  minus  selections  and  intermediate  group =0.155  ±0.133. 


GENETICS  7:    Mr  1922 


INFORMATION  FOR  CONTRIBUTORS 

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GENETICS,  MARCH  1922 


TABLE  OF  CONTENTS 


PAGE 


MACDOWELL,  E.  C.,  The  influence  of  alcohol  on  the  fertility  of  white 


rats 


117 

LA  RUE,  CARL  DOWNEY,  The  results  of  selection  within  pure  lines  of 

PestalozziaGuefini  Desm 142 


THE  GALTON  AND  MENDEL  CENTENARY 

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